Multiplex branched-chain dna assays

0001This application is a non-provisional utility patent application claiming priority to and benefit of the following prior provisional patent application: <patcit id="pcit0001" dnum="USSN60680976A"><text>USSN 60/680,976, filed May 12, 2005</text></patcit>, entitled "MULTIPLEX BRANCHED-CHAIN DNA ASSAYS" by Luo and Yang.

FIELD OF THE INVENTION

0002The present invention is in the field of nucleic acid detection. The invention includes methods for detecting the presence of 10 or more nucleic acids simultaneously in a single sample. The invention also includes compositions and kits related to the methods.

BACKGROUND OF THE INVENTION

0003Global gene expression profiling and other technologies have identified a large number of genes whose expression is altered, e.g., in diseased tissues or in tissues and cells treated with pharmaceutical agents (<nplcit id="ncit0001" npl-type="s"><text>Lockhart and Winzeler (2000) "Genomics, gene expression and DNA arrays" Nature 405:827-36</text></nplcit> and <nplcit id="ncit0002" npl-type="s"><text>Gunther et al. (2003) "Prediction of clinical drug efficacy by classification of drug-induced genomic expression profiles in vitro" Proc Natl Acad Sci USA 100:9608-13</text></nplcit>). Such genes are being increasingly used as biomarkers in disease diagnosis, staging, and prognosis (<nplcit id="ncit0003" npl-type="s"><text>Golub et al. (1999) "Molecular classification of cancer: class discovery and class prediction by gene expression monitoring" Science 286:531-7</text></nplcit>); target identification, validation and pathway analysis (<nplcit id="ncit0004" npl-type="s"><text>Roberts et al. (2000) "Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles" Science 287:873-80</text></nplcit>); drug screening (<nplcit id="ncit0005" npl-type="s"><text>Hamadeh et al. (2002) "Prediction of compound signature using high density gene expression profiling" Toxicol Sci 67:232-40</text></nplcit>); and studies of drug efficacy, structure-activity relationship, toxicity, and drug-target interactions (<nplcit id="ncit0006" npl-type="s"><text>Gerhold et al. (2001) "Monitoring expression of genes involved in drug metabolism and toxicology using DNA microarrays" Physiol Genomics 5:161-70</text></nplcit> and <nplcit id="ncit0007" npl-type="s"><text>Thomas et al. (2001) "Identification of toxicologically predictive gene sets using cDNA microarrays" Mol Pharmacol 60:1189-94</text></nplcit>). As biomarkers are identified, their involvement in disease management and drug development will need to be evaluated in higher throughput and broader populations of samples. Simpler and more flexible expression profiling technology that allows the expression analysis of multiple genes with higher data quality and higher throughput is therefore needed.

0004Levels of RNA expression have traditionally been measured using Northern blot and nuclease protection assays. However, these approaches are time-consuming and have limited sensitivity, and the data generated are more qualitative than quantitative in nature. Greater sensitivity and quantification is possible with reverse transcription polymerase chain reaction (RT-PCR) based methods, such as quantitative real-time RT-PCR, but these approaches have low multiplex capabilities (<nplcit id="ncit0008" npl-type="s"><text>Bustin (2002) "Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems" J Mol Endocrinol 29:23-39</text></nplcit> and <nplcit id="ncit0009" npl-type="s"><text>Bustin and Nolan (2004) "Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction" J Biomol Tech. 15:155-66</text></nplcit>). Microarray technology has been widely used in discovery research, but its moderate sensitivity and its relatively long experimental procedure have limited its use in high throughput expression profiling applications (<nplcit id="ncit0010" npl-type="s"><text>Epstein and Butow (2000) "Microarray technology - enhanced versatility, persistent challenge" Curr Opin Biotechnol. 11:36-41</text></nplcit>).

0005Most of the current methods of mRNA quantification require RNA isolation, reverse transcription, and target amplification, each of which introduces variability that leads to low overall assay precision. Recently, a multiplex screening assay for mRNA quantification combining nuclease protection with luminescent array detection was reported (<nplcit id="ncit0011" npl-type="s"><text>Martel et al. (2002) "Multiplexed screening assay for mRNA combining nuclease protection with luminescent array detection" Assay Drug Dev Technol. 1:61-71</text></nplcit>). Although this assay has the advantage of measuring mRNA transcripts directly from cell lysates, limited assay sensitivity and reproducibility were reported. Another multiplex mRNA assay without the need for RNA isolation was also reported (<nplcit id="ncit0012" npl-type="s"><text>Tian et al. (2004) "Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis" Nucleic Acids Res. 32:e126</text></nplcit>). This assay couples the primary Invader® mRNA assay with small fluorescent molecule eTags that can be distinguished by capillary electrophoresis through distinct charge-to-mass ratios of eTags. However, this assay requires the use of a specially designed and synthesized set of eTagged signal probes, complicated capillary electrophoresis equipment, and a special data analysis package.

0006Among other aspects, the present invention provides methods that overcome the above noted limitations and permit rapid, simple, and sensitive detection of multiple mRNAs (and/or other nucleic acids) simultaneously. A complete understanding of the invention will be obtained upon review of the following.

SUMMARY OF THE INVENTION

0007In one aspect, the present invention provides methods of detecting 10 or more nucleic acids of interest in a multiplex branched-chain DNA assay. Different nucleic acids are captured through cooperative hybridization events on different, identifiable subsets of particles or at different selected positions on a spatially addressable solid support. Compositions and kits related to the methods are also provided.

0008A first general class of embodiments provides methods of detecting 10 or more nucleic acids of interest. In the methods, a sample, a pooled population of particles, and 10 or more subsets of n capture extenders, wherein n is at least two, are provided. The sample comprises or is suspected of comprising the nucleic acids of interest. The pooled population of particles includes 10 or more subsets of particles, and a plurality of the particles in each subset are distinguishable from a plurality of the particles in every other subset. The particles in each subset have associated therewith a different capture probe. Each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected subset of the particles. Each nucleic acid of interest can thus, by hybridizing to its corresponding subset of n capture extenders which are in turn hybridized to a corresponding capture probe, be associated with an identifiable subset of the particles.

0009The sample, the pooled population of particles, and the subsets of n capture extenders are contacted, any nucleic acid of interest present in the sample is hybridized to its corresponding subset of n capture extenders, and the subset of n capture extenders is hybridized to its corresponding capture probe. The hybridizing the nucleic acid of interest to the n capture extenders and the n capture extenders to the corresponding capture probe captures the nucleic acid on the subset of particles with which the capture extenders are associated. The hybridizing the subset of n capture extenders to the corresponding capture probe is performed at a hybridization temperature which is greater than a melting temperature T_m of a complex between each individual capture extender and its corresponding capture probe.

0010To determine which subsets of particles have a nucleic acid of interest captured on the particles, one or more label extenders and a label probe system comprising a label are hybridized to any nucleic acid of interest captured on the particles, and at least a portion of the particles from each subset are identified and the presence or absence of the label is detected on those particles. Since a correlation exists between a particular subset of particles and a particular nucleic acid of interest, which subsets of particles have the label present indicates which of the nucleic acids of interest were present in the sample.

0011The methods are useful for multiplex detection of nucleic acids, optionally highly multiplex detection. Thus, the 10 or more nucleic acids of interest (i.e., the nucleic acids to be detected) optionally comprise 20 or more, 30 or more, 40 or more, 50 or more, or even 100 or more nucleic acids of interest. A like number of subsets of particles and subsets of capture extenders are provided; thus, the or more subsets of particles can comprise 20 or more, 30 or more, 40 or more, 50 or more, or even 100 or more subsets of particles, while the 10 or more subsets of n capture extenders can comprise 20 or more, 30 or more, 40 or more, 50 or more, or even 100 or more subsets of n capture extenders.

0012In one class of embodiments, the particles are microspheres. The microspheres of each subset can be distinguishable from those of the other subsets, e.g., on the basis of their fluorescent emission spectrum, their diameter, or a combination thereof.

0013As noted, each of the 10 or more subsets of capture extenders includes n capture extenders, where n is at least two. Preferably, n is at least three, and n can be at least four or at least five or more. Typically, but not necessarily, n is at most ten. The n capture extenders in a subset hybridize to nonoverlapping polynucleotide sequences in the corresponding nucleic acid of interest. The nonoverlapping polynucleotide sequences can, but need not be, consecutive within the nucleic acid of interest.

0014Each capture extender is capable of hybridizing to its corresponding capture probe. The capture extender typically includes a polynucleotide sequence C-1 that is complementary to a polynucleotide sequence C-2 in its corresponding capture-probe. In one aspect, C-1 and C-2 are 20 nucleotides or less in length. In one class of embodiments, C-1 and C-2 are between 9 and 17 nucleotides in length (inclusive), preferably between 12 and 1S nucleotides (inclusive).

0015As noted, the hybridizing the subset of n capture extenders to the corresponding capture probe is performed at a hybridization temperature which is greater than a melting temperature T_m of a complex between each individual capture extender and its corresponding capture probe. The hybridization temperature is typically about 5°C or more greater than the T_m, e.g., about 7°C or more, about 10°C or more, about 12°C or more, about 15°C or more, about 17°C or more, or even about 20°C or more greater than the T_m.

0016In one class of embodiments, contacting the sample, the pooled population of particles, and the subsets of n capture extenders comprises combining the sample with the subsets of n capture extenders to form a mixture, and then combining the mixture with the pooled population of particles.

0017In a preferred class of embodiments, hybridizing one or more label extenders and a label probe system to any nucleic acid of interest captured on the particles comprises providing 10 or more subsets of one or more label extenders, wherein each subset of label extenders is capable of hybridizing to one of the nucleic acids of interest, hybridizing any nucleic acid of interest captured on the particles to its corresponding subset of label extenders, and hybridizing the label probe system to the label extenders. The 10 or more subsets of label extenders can include, e.g., 20 or more, 30 or more, 40 or more, 50 or more, or even 100 or more subsets of label extenders. The hybridizations can be performed simultaneously or sequentially, in essentially any order. In one embodiment, any nucleic acid of interest present in the sample is hybridized to its corresponding subset of label extenders and to its corresponding subset of n capture extenders, then the subset of n capture extenders is hybridized to its corresponding capture probe.

0018The label probe system optionally includes an amplification multimer and a plurality of label probes, wherein the amplification multimer is capable of hybridizing to a label extender and to a plurality of label probes. In one class of embodiments, the label probe comprises the label. In one aspect, the label is a fluorescent label, and detecting the presence of the label on the particles comprises detecting a fluorescent signal from the label.

0019The methods can optionally be used to quantitate the amounts of the nucleic acids of interest present in the sample. For example, in one class of embodiments, an intensity of a signal from the label is measured, e.g., for each subset of particles, and correlated with a quantity of the corresponding nucleic acid of interest present.

0020At any of various steps, materials not captured on the particles are optionally separated from the particles. For example, after the capture extenders, nucleic acids, label extenders, blocking probes, and particle-bound capture probes are hybridized, the particles are optionally washed to remove unbound nucleic acids and probes; after the label extenders and amplification multimer are hybridized, the particles are optionally washed to remove unbound amplification multimer; and/or after the label probes are hybridized to the amplification multimer, the particles are optionally washed to remove unbound label probe prior to detection of the label.

0021The methods can be used to detect the presence of the nucleic acids of interest in essentially any type of sample. For example, the sample can be derived from an animal, a human, a plant, a cultured cell, a virus, a bacterium, a pathogen, and/or a microorganism. The sample optionally includes a cell lysate, an intercellular fluid, a bodily fluid (including, but not limited to, blood, serum, saliva, urine, sputum, or spinal fluid), and/or a conditioned culture medium, and is optionally derived from a tissue (e.g., a tissue homogenate), a biopsy, and/or a tumor. Similarly, the nucleic acids can be essentially any desired nucleic acids. As just a few examples, the nucleic acids of interest can be derived from one or more of an animal, a human, a plant, a cultured cell, a microorganism, a virus, a bacterium, or a pathogen. In one class of embodiments, the 10 or more nucleic acids of interest comprise 10 or more mRNAs.

0022In one class of embodiments, at least one of the nucleic acids of interest is present in the sample in a non-zero amount of 200 amol or less, 150 amol or less, 100 amol or less, 50 amol or less, 10 amol or less, 1 amol or less, or even 0.1 amol or less. In one exemplary class of embodiments, the sample includes a first nucleic acid of interest, and at least 30%, at least 50%, at least 80%, at least 90%, at least 95%, or even at least 99% of a total amount of the first nucleic acid present in the sample is captured on a first subset of particles. Second, third, etc. nucleic acids can similarly be quantitatively captured. Such quantitative capture can occur without capture of a significant amount of undesired nucleic acids, even those of very similar sequence to the nucleic acid of interest.

0023Thus, in one class of embodiments, the sample comprises or is suspected of comprising a first nucleic acid of interest and a second nucleic acid which has a polynucleotide sequence which is 95% or more identical to that of the first nucleic acid (e.g., 96% or more, 97% or more, 98% or more, or even 99% or more identical). The first nucleic acid, if present in the sample, is captured on a first subset of particles, while the . second nucleic acid comprises 1% or less of a total amount of nucleic acid captured on the first subset of particles (e.g., 0.5% or less, 0.2% or less, or even 0.1% or less).

0024As just one example of how closely related nucleic acids can be distinguished using the methods of the invention, different splice variants of a given mRNA can be distinguished. Thus, in one class of embodiments, the sample comprises a first nucleic acid of interest and a second nucleic acid, where the first nucleic acid is a first splice variant and the second nucleic acid is a second splice variant of the given mRNA. A first subset of n capture extenders is capable of hybridizing to the first splice variant, of which at most n-1 capture extenders are capable of hybridizing to the second splice variant. Preferably, hybridization of the n capture extenders to the first splice variant captures the first splice variant on a first subset of particles while hybridization of the at most n-1 capture extenders to the second splice variant does not capture the second splice variant on the first subset of particles.

0025Another general class of embodiments provides a composition for detecting 10 or more nucleic acids of interest that includes 10 or more subsets of particles, 10 or more subsets of n capture extenders, wherein n is at least two, 10 or more subsets of one or more label extenders, and a label probe system comprising a label. A plurality of the particles in each subset are distinguishable from a plurality of the particles in every other subset. The particles in each subset have associated therewith a different capture probe. Each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected subset of the particles. When the nucleic acid of interest corresponding to a subset of n capture extenders is present in the composition and is hybridized to the subset of n capture extenders, which are hybridized to the corresponding capture probe, the nucleic acid of interest is hybridized to the subset of n capture extenders at a hybridization temperature which is greater than a melting temperature T_m of a complex between each individual capture extender and the capture probe.

0026Essentially all of the features noted for the embodiments above apply to these embodiments as well, as relevant; for example, with respect to number of capture extenders per subset, composition of the label probe system, type of label, inclusion of blocking probes, configuration of the capture extenders, capture probes, label extenders, and/or blocking probes, number of nucleic acids of interest and of subsets of particles, capture extenders and label extenders, source of the sample and/or nucleic acids, and/or the like.

0027A related general class of embodiments provides a composition comprising 10 or more subsets of particles, 10 or more subsets of n capture extenders, wherein n is at least two, 10 or more subsets of one or more label extenders, a label probe system comprising a label, and at least a first nucleic acid of interest. A plurality of the particles in each subset are distinguishable from a plurality of the particles in every other subset. The particles in each subset have associated therewith a different capture probe. Each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected subset of the particles. In this class of embodiments, the composition is maintained at a hybridization temperature, which hybridization temperature is greater than a melting temperature T_m of a complex between each individual capture extender and its corresponding capture probe. The first nucleic acid of interest is hybridized to a first subset of n first capture extenders, which first capture extenders are hybridized to a first capture probe.

0028Essentially all of the features noted for the embodiments above apply to these embodiments as well, as relevant; for example, with respect to number of capture extenders per subset, composition of the label probe system, type of label, inclusion of blocking probes, configuration of the capture extenders, capture probes, label extenders, and/or blocking probes, number of nucleic acids of interest and of subsets of particles, capture extenders and label extenders, source of the sample and/or nucleic acids, and/or the like.

0029Discussed herein is a kit for detecting two or more nucleic acids of interest. The kit includes two or more subsets of particles, two or more subsets of n capture extenders, wherein n is at least two, two or more subsets of one or more label extenders, and a label probe system comprising a label, packaged in one or more containers. A plurality of the particles in each subset are distinguishable from a plurality of the particles in every other subset. The particles in each subset have associated therewith a different capture probe. Each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected subset of the particles. When the nucleic acid of interest corresponding to a subset of n capture extenders is hybridized to the subset of n capture extenders, which are hybridized to the corresponding capture probe, the nucleic acid of interest is hybridized to the subset of n capture extenders at a hybridization temperature which is greater than a melting temperature T_m of a complex between each individual capture extender and the capture probe. The kit optionally also includes instructions for using the kit to capture and detect the nucleic acids of interest, one or more buffered solutions (e.g., lysis buffer, diluent, hybridization buffer, and/or wash buffer), standards comprising one or more nucleic acids at known concentration, and/or the like.

0030Essentially all of the features noted for the embodiments above apply to the kits as well, as relevant; for example, with respect to number of capture extenders per subset, composition of the label probe system, type of label, inclusion of blocking probes, configuration of the capture extenders, capture probes, label extenders, and/or blocking probes, number of nucleic acids of interest and of subsets of particles, capture extenders and label extenders, source of the sample and/or nucleic acids, and/or the like.

0031Yet another general class of embodiments includes methods of detecting 10 or more nucleic acids of interest. In the methods, a sample, a solid support, and 10 or more subsets of n capture extenders, wherein n is at least two, are provided. The sample comprises or is suspected of comprising the nucleic acids of interest. The solid support comprises 10 or more capture probes, each of which is provided at a selected position on the solid support. Each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected position on the solid support. Each nucleic acid of interest can thus, by hybridizing to its corresponding subset of n capture extenders which are in turn hybridized to a corresponding capture probe, be associated with, e.g., a known, predetermined location on the solid support. The sample, the solid support, and the subsets of n capture extenders are contacted, any nucleic acid of interest present in the sample is hybridized to its corresponding subset of n capture extenders, and the subset of n capture extenders is hybridized to its corresponding capture probe. The hybridizing the nucleic acid of interest to the n capture extenders and the n capture extenders to the corresponding capture probe captures the nucleic acid on the solid support at the selected position with which the capture extenders are associated. To determine which positions on the solid support have a nucleic acid of interest captured at that position, one or more label extenders and a label probe system comprising a label are hybridized to any nucleic acid of interest captured on the solid support, and the presence or absence of the label at the selected positions on the solid support is detected. Since a correlation exists between a particular position on the support and a particular nucleic acid of interest, which positions have a label present indicates which of the nucleic acids of interest were present in the sample.

0032Hybridizing the subset of n capture extenders to the corresponding capture probe is optionally performed at a hybridization temperature which is greater than a melting temperature T_m of a complex between each individual capture extender and its corresponding capture probe.

0033Essentially all of the features noted for the embodiments above apply to these embodiments as well, as relevant; for example, with respect to number of capture extenders per subset, composition of the label probe system, type of label, inclusion of blocking probes, configuration of the capture extenders, capture probes, label extenders, and/or blocking probes, number of nucleic acids of interest and of selected positions on the solid support, capture extenders and label extenders, source of the sample and/or nucleic acids, and/or the like.

0034Another general class of embodiments provides a composition for detecting 10 or more nucleic acids of interest, the composition that includes a solid support comprising 10 or more capture probes, each of which is provided at a selected position on the solid support, 10 or more subsets of n capture extenders, wherein n is at least two, or more subsets of one or more label extenders, and a label probe system comprising a label. Each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected position on the solid support.

0035Essentially all of the features noted for the embodiments above apply to these embodiments as well, as relevant; for example, with respect to number of capture extenders per subset, composition of the label probe system, type of label, inclusion of blocking probes, configuration of the capture extenders, capture probes, label extenders, and/or blocking probes, number of nucleic acids of interest and of selected positions on the solid support, capture extenders and label extenders, source of the sample and/or nucleic acids, and/or the like.

0036Discussed herein is a kit for detecting two or more nucleic acids of interest. The kit includes a solid support comprising two or more capture probes, each of which is provided at a selected position on the solid support, two or more subsets of n capture extenders, wherein n is at least two, two or more subsets of one or more label extenders, and a label probe system comprising a label, packaged in one or more containers. Each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected position on the solid support. The kit optionally also includes instructions for using the kit to capture and detect the nucleic acids of interest, one or more buffered solutions (e.g., lysis buffer, diluent, hybridization buffer, and/or wash buffer), standards comprising one or more nucleic acids at known concentration, and/or the like.

0037Essentially all of the features noted for the embodiments above apply to these kits as well, as relevant; for example, with respect to number of capture extenders per subset, composition of the label probe system, type of label, inclusion of blocking probes, configuration of the capture extenders, capture probes, label extenders, and/or blocking probes, number of nucleic acids of interest and of selected positions on the solid support, capture extenders and label extenders, source of the sample and/or nucleic acids, and/or the like.

BRIEF DESCRIPTION OF THE DRAWINGS

0038<figref idref="f0001"><b>Figure 1</b></figref> schematically illustrates a typical standard bDNA assay.

0039<figref idref="f0002 f0003"><b>Figure 2</b></figref><b>Panels A-E</b> schematically depict a multiplex bDNA assay, in which the nucleic acids of interest are captured on distinguishable subsets of microspheres and then detected.

0040<figref idref="f0004"><b>Figure 3</b></figref> schematically depicts an exemplary embodiment in which two splice variants are specifically captured on distinguishable subsets of microspheres and then detected.

0041<figref idref="f0005 f0006"><b>Figure 4</b></figref><b>Panels A-D</b> schematically depict a multiplex bDNA assay, in which the nucleic acids of interest are captured at selected positions on a solid support and then detected. <b>Panel A</b> shows a top view of the solid support, while <b>Panels B-D</b> show the support in cross-section.

0042<figref idref="f0007"><b>Figure 5</b></figref><b>Panels A-C</b> schematically depict an overview of a multiplex bDNA assay.

0043<figref idref="f0008"><b>Figure 6</b></figref><b>Panel A</b> schematically depicts simple hybridization of a single CE to a CP and a target mRNA. <b>Panel B</b> schematically depicts cooperative hybridization of CEs to a CP and a target mRNA. <b>Panel C</b> depicts a bar graph illustrating simple hybridization between a CE and CP under standard bDNA assay conditions. <b>Panel D</b> depicts a line graph illustrating cooperative hybridization between CEs and CPs under standard bDNA assay conditions.

0044<figref idref="f0009 f0010"><b>Figure 7</b></figref><b>Panel A</b> depicts a graph illustrating evaluation of the cytokine panel for cross-reactivity. Individual mRNA transcript names are presented on the x-axis, bead subsets are identified by number on the y-axis, and fluorescent signal is graphed on the z-axis. <b>Panel B</b> depicts a line graph of fluorescent intensity observed for a dilution series of dapB transcript in the presence (squares) or absence (diamonds) of 0.2 µg total RNA, for evaluation of specificity. <b>Panel C</b> depicts a line graph of fluorescent intensity observed for a dilution series of IL10 transcript in the presence (squares) or absence (diamonds) of 0.2 µg total RNA, for evaluation of specificity. <b>Panel D</b> depicts a line graph of fluorescent intensity for a dilution series of all the transcripts in the cytokine panel, for evaluation of assay sensitivity and dynamic range.

0045<figref idref="f0011"><b>Figure 8</b></figref><b>Panel A</b> depicts a bar graph illustrating induction of cytokine gene expression upon PMA/LPS treatment of U937 cells, as detected by a 10-plex bDNA assay. <b>Panel B</b> depicts a bar graph illustrating induction of cytokine gene expression upon PMA/LPS treatment of U937 cells, as detected by single-plex bDNA assays using the same cell lysates.

0046<figref idref="f0012 f0013"><b>Figure 9</b></figref><b>Panel A</b> depicts bar graphs illustrating time-dependent expression of pro- and anti-apoptotic genes upon TNFα treatment of HeLa cells, as detected by a 10-plex bDNA assay. <b>Panel B</b> depicts bar graphs illustrating expression of pro- and anti-apoptotic genes in untreated cells and in TNFα-treated cells, 3 hours after treatment, as detected by single-plex bDNA assays (top) and a 10-plex bDNA assay (bottom).

0047Schematic figures are not necessarily to scale.

DEFINITIONS

0048Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

0049As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" includes a plurality of such molecules, and the like.

0050The term "about" as used herein indicates the value of a given quantity varies by +/-10% of the value, or optionally +/- 5% of the value, or in some embodiments, by +/-1 % of the value so described.

0051The term "polynucleotide" (and the equivalent term "nucleic acid") encompasses any physical string of monomer units that can be corresponded to a string of nucleotides, including a polymer of nucleotides (e.g., a typical DNA or RNA polymer), peptide nucleic acids (PNAs), modified oligonucleotides (e.g., oligonucleotides comprising nucleotides that are not typical to biological RNA or DNA, such as 2'-O-methylated oligonucleotides), and the like. The nucleotides of the polynucleotide can be deoxyribonucleotides, ribonucleotides or nucleotide analogs, can be natural or non-natural, and can be unsubstituted, unmodified, substituted or modified. The nucleotides can be linked by phosphodiester bonds, or by phosphorothioate linkages, methylphosphonate linkages, boranophosphate linkages, or the like. The polynucleotide can additionally comprise non-nucleotide elements such as labels, quenchers, blocking groups, or the like. The polynucleotide can be, e.g., single-stranded or double-stranded.

0052A "polynucleotide sequence" or "nucleotide sequence" is a polymer of nucleotides (an oligonucleotide, a DNA, a nucleic acid, etc.) or a character string representing a nucleotide polymer, depending on context. From any specified polynucleotide sequence, either the given nucleic acid or the complementary polynucleotide sequence (e.g., the complementary nucleic acid) can be determined.

0053Two polynucleotides "hybridize" when they associate to form a stable duplex, e.g., under relevant assay conditions. Nucleic acids hybridize due to a variety of well characterized physico-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. An extensive guide to the hybridization of nucleic acids is found in <nplcit id="ncit0013" npl-type="b"><text>Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part I chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays" (Elsevier, New York), as well as in Ausubel</text></nplcit>, <i>infra.</i>

0054The "T_m" (melting temperature) of a nucleic acid duplex under specified conditions (e.g., relevant assay conditions) is the temperature at which half of the base pairs in a population of the duplex are disassociated and half are associated. The T_m for a particular duplex can be calculated and/or measured, e.g., by obtaining a thermal denaturation curve for the duplex (where the T_m is the temperature corresponding to the midpoint in the observed transition from double-stranded to single-stranded form).

0055The term "complementary" refers to a polynucleotide that forms a stable duplex with its "complement," e.g., under relevant assay conditions. Typically, two polynucleotide sequences that are complementary to each other have mismatches at less than about 20% of the bases, at less than about 10% of the bases, preferably at less than about 5% of the bases, and more preferably have no mismatches.

0056A "capture extender" or "CE" is a polynucleotide that is capable of hybridizing to a nucleic acid of interest and to a capture probe. The capture extender typically has a first polynucleotide sequence C-1, which is complementary to the capture probe, and a second polynucleotide sequence C-3, which is complementary to a polynucleotide sequence of the nucleic acid of interest. Sequences C-1 and C-3 are typically not complementary to each other. The capture extender is preferably single-stranded.

0057A "capture probe" or "CP" is a polynucleotide that is capable of hybridizing to at least one capture extender and that is tightly bound (e.g., covalently or noncovalently, directly or through a linker, e.g., streptavidin-biotin or the like) to a solid support, a spatially addressable solid support, a slide, a particle, a microsphere, or the like. The capture probe typically comprises at least one polynucleotide sequence C-2 that is complementary to polynucleotide sequence C-1 of at least one capture extender. The capture probe is preferably single-stranded.

0058A "label extender" or "LE" is a polynucleotide that is capable of hybridizing to a nucleic acid of interest and to a label probe system. The label extender typically has a first polynucleotide sequence L-1, which is complementary to a polynucleotide sequence of the nucleic acid of interest, and a second polynucleotide sequence L-2, which is complementary to a polynucleotide sequence of the label probe system (e.g., L-2 can be complementary to a polynucleotide sequence of an amplification multimer, a preamplifier, a label probe, or the like). The label extender is preferably single-stranded.

0059A "label" is a moiety that facilitates detection of a molecule. Common labels in the context of the present invention include fluorescent, luminescent, light-scattering, and/or colorimetric labels. Suitable labels include enzymes and fluorescent moieties, as well as radionuclides, substrates, cofactors, inhibitors, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include <patcit id="pcit0002" dnum="US3817837A"><text>U.S. Pat. Nos. 3,817,837</text></patcit>; <patcit id="pcit0003" dnum="US3850752A"><text>3,850,752</text></patcit>; <patcit id="pcit0004" dnum="US3939350A"><text>3,939,350</text></patcit>; <patcit id="pcit0005" dnum="US3996345A"><text>3,996,345</text></patcit>; <patcit id="pcit0006" dnum="US4277437A"><text>4,277,437</text></patcit>; <patcit id="pcit0007" dnum="US4275149A"><text>4,275,149</text></patcit>; and <patcit id="pcit0008" dnum="US4366241A"><text>4,366,241</text></patcit>. Many labels are commercially available and can be used in the context of the invention.

0060A "label probe system" comprises one or more polynucleotides that collectively comprise a label and a polynucleotide sequence M-1, which is capable of hybridizing to at least one label extender. The label provides a signal, directly or indirectly. Polynucleotide sequence M-1 is typically complementary to sequence L-2 in the label extenders. The label probe system can include a plurality of label probes (e.g., a plurality of identical label probes) and an amplification multimer; it optionally also includes a preamplifier or the like, or optionally includes only label probes, for example.

0061An "amplification multimer" is a polynucleotide comprising a plurality of polynucleotide sequences M-2, typically (but not necessarily) identical polynucleotide sequences M-2. Polynucleotide sequence M-2 is complementary to a polynucleotide sequence in the label probe. The amplification multimer also includes at least one polynucleotide sequence that is capable of hybridizing to a label extender or to a nucleic acid that hybridizes to the label extender, e.g., a preamplifier. For example, the amplification multimer optionally includes at least one polynucleotide sequence M-1; polynucleotide sequence M-1 is typically complementary to polynucleotide sequence L-2 of the label extenders. Similarly, the amplification multimer optionally includes at least one polynucleotide sequence that is complementary to a polynucleotide sequence in a preamplifier. The amplification multimer can be, e.g., a linear or a branched nucleic acid. As noted for all polynucleotides, the amplification multimer can include modified nucleotides and/or nonstandard internucleotide linkages as well as standard deoxyribonucleotides, ribonucleotides, and/or phosphodiester bonds. Suitable amplification multimers are described, for example, in USPN 5,635,352, USPN 5,124,246, USPN 5,710,264, and USPN 5,849,481.

0062A "label probe" or "LP" is a single-stranded polynucleotide that comprises a label (or optionally that is configured to bind to a label) that directly or indirectly provides a detectable signal. The label probe typically comprises a polynucleotide sequence that is complementary to the repeating polynucleotide sequence M-2 of the amplification multimer; however, if no amplification multimer is used in the bDNA assay, the label probe can, e.g., hybridize directly to a label extender.

0063A "preamplifier" is a nucleic acid that serves as an intermediate between at least one label extender and amplification multimer. Typically, the preamplifier is capable of hybridizing simultaneously to at least one label extender and to a plurality of amplification multimers.

0064A "microsphere" is a small spherical, or roughly spherical, particle. A microsphere typically has a diameter less than about 1000 micrometers (e.g., less than about 100 micrometers, optionally less than about 10 micrometers).

0065A "microorganism" is an organism of microscopic or submicroscopic size. Examples include, but are not limited to, bacteria, fungi, yeast, protozoans, microscopic algae (e.g., unicellular algae), viruses (which are typically included in this category although they are incapable of growth and reproduction outside of host cells), subviral agents, viroids, and mycoplasma.

0066A variety of additional terms are defined or otherwise characterized herein.

DETAILED DESCRIPTION

0067The present invention provides methods and compositions for multiplex detection of nucleic acids. Nucleic acids of interest are captured to distinct subsets of particles or to selected positions on a solid support through cooperative hybridization, and they are then detected in a branched-chain DNA assay.

0068Branched-chain DNA (bDNA) signal amplification technology has been used, e.g., to detect and quantify mRNA transcripts in cell lines and to determine viral loads in blood. The bDNA assay is a sandwich nucleic acid hybridization procedure that enables direct measurement of mRNA expression, e.g., from crude cell lysate. It provides direct quantification of nucleic acid molecules at physiological levels. Several advantages of the technology distinguish it from other DNA/RNA amplification technologies, including linear amplification, good sensitivity and dynamic range, great precision and accuracy, simple sample preparation procedure, and reduced sample-to-sample variation.

0069In brief, in a typical bDNA assay for gene expression analysis (<figref idref="f0001"><b>Figure 1</b></figref>), a target mRNA whose expression is to be detected is released from cells and captured by a Capture Probe (CP) on a solid surface (e.g., a well of a microtiter plate) through synthetic oligonucleotide probes called Capture Extenders (CEs). Each capture extender has a first polynucleotide sequence that can hybridize to the target mRNA and a second polynucleotide sequence that can hybridize to the capture probe. Typically, two or more capture extenders are used. Probes of another type, called Label Extenders (LEs), hybridize to different sequences on the target mRNA and to sequences on an amplification multimer. Additionally, Blocking Probes (BPs) are often used to reduce non-specific target probe binding. A probe set for a given mRNA thus consists of CEs, LEs, and optionally BPs for the target mRNA. The CEs, LEs, and BPs are complementary to nonoverlapping sequences in the target mRNA, and are typically, but not necessarily, contiguous.

0070Signal amplification begins with the binding of the LEs to the target mRNA. An amplification multimer is then typically hybridized to the LEs. The amplification multimer has multiple copies of a sequence that is complementary to a label probe (it is worth noting that the amplification multimer is typically, but not necessarily, a branched-chain nucleic acid; for example, the amplification multimer can be a branched, forked, or comb-like nucleic acid or a linear nucleic acid). A label, for example, alkaline phosphatase, is covalently attached to each label probe. (Alternatively, the label can be noncovalently bound to the label probes.) In the final step, labeled complexes are detected, e.g., by the alkaline phosphatase-mediated degradation of a chemilumigenic substrate, e.g., dioxetane. Luminescence is reported as relative light unit (RLUs) on a microplate reader. The amount of chemiluminescence is proportional to the level of mRNA expressed from the target gene.

0071In the preceding example, the amplification multimer and the label probes comprise a label probe system. In another example, the label probe system also comprises a preamplifier, e.g., as described in USPN 5,635,352 and USPN 5,681,697, which further amplifies the signal from a single target mRNA. In yet another example, the label extenders hybridize directly to the label probes and no amplification multimer or preamplifier is used, so the signal from a single target mRNA molecule is only amplified by the number of distinct label extenders that hybridize to that mRNA.

0072Basic bDNA assays have been well described. See, e.g., <patcit id="pcit0009" dnum="USPN4868105A"><text>USPN 4,868,105 to Urdea et al.</text></patcit> entitled "Solution phase nucleic acid sandwich assay"; <patcit id="pcit0010" dnum="USPN5635352A"><text>USPN 5,635,352 to Urdea et al.</text></patcit> entitled "Solution phase nucleic acid sandwich assays having reduced background noise"; <patcit id="pcit0011" dnum="USPN5681697A"><text>USPN 5,681,697 to Urdea et al.</text></patcit> entitled "Solution phase nucleic acid sandwich assays having reduced background noise and kits therefor"; <patcit id="pcit0012" dnum="USPN5124246A"><text>USPN 5,124,246 to Urdea et al.</text></patcit> entitled "Nucleic acid multimers and amplified nucleic acid hybridization assays using same"; <patcit id="pcit0013" dnum="USPN5624802A"><text>USPN 5,624,802 to Urdea et al.</text></patcit> entitled "Nucleic acid multimers and amplified nucleic acid hybridization assays using same"; <patcit id="pcit0014" dnum="USPN5849481A"><text>USPN 5,849,481 to Urdea et al.</text></patcit> entitled "Nucleic acid hybridization assays employing large comb-type branched polynucleotides"; <patcit id="pcit0015" dnum="USPN5710264A"><text>USPN 5,710,264 to Urdea et al.</text></patcit> entitled "Large comb type branched polynucleotides"; <patcit id="pcit0016" dnum="USPN5594118A"><text>USPN 5,594,118 to Urdea and Horn</text></patcit> entitled "Modified N-4 nucleotides for use in amplified nucleic acid hybridization assays"; <patcit id="pcit0017" dnum="USPN5093232A"><text>USPN 5,093,232 to Urdea and Horn</text></patcit> entitled "Nucleic acid probes"; <patcit id="pcit0018" dnum="USPN4910300A"><text>USPN 4,910,300 to Urdea and Horn</text></patcit> entitled "Method for making nucleic acid probes"; <patcit id="pcit0019" dnum="USPN5359100A"><text>USPN 5,359,100</text></patcit>; <patcit id="pcit0020" dnum="USPN5571670A"><text>USPN 5,571,670</text></patcit>; <patcit id="pcit0021" dnum="USPN5614362A"><text>USPN 5,614,362</text></patcit>; <patcit id="pcit0022" dnum="USPN6235465A"><text>USPN 6,235,465</text></patcit>; <patcit id="pcit0023" dnum="USPN5712383A"><text>USPN 5,712,383</text></patcit>; <patcit id="pcit0024" dnum="USPN5747244A"><text>USPN 5,747,244</text></patcit>; <patcit id="pcit0025" dnum="USPN6232462A"><text>USPN 6,232,462</text></patcit>; <patcit id="pcit0026" dnum="USPN5681702A"><text>USPN 5,681,702</text></patcit>; <patcit id="pcit0027" dnum="USPN5780610A"><text>USPN 5,780,610</text></patcit>; <patcit id="pcit0028" dnum="USPN5780227A"><text>USPN 5,780,227 to Sheridan et al.</text></patcit> entitled "Oligonucleotide probe conjugated to a purified hydrophilic alkaline phosphatase and uses thereof"; U.S. patent application Publication No. <patcit id="pcit0029" dnum="US2002172950A"><text>US2002172950 by Kenny et al.</text></patcit> entitled "Highly sensitive gene detection and localization using in situ branched-DNA hybridization"; <nplcit id="ncit0014" npl-type="s"><text>Wang et al. (1997) "Regulation of insulin preRNA splicing by glucose" Proc Nat Acad Sci USA 94:4360-4365</text></nplcit>; <nplcit id="ncit0015" npl-type="b"><text>Collins et al. (1998) "Branched DNA (bDNA) technology for direct quantification of nucleic acids: Design and performance" in Gene Quantification, F Ferre, ed</text></nplcit>.; and <nplcit id="ncit0016" npl-type="s"><text>Wilber and Urdea (1998) "Quantification of HCV RNA in clinical specimens by branched DNA (bDNA) technology" Methods in Molecular Medicine: Hepatitis C 19:71-78</text></nplcit>. In addition, kits for performing basic bDNA assays (QuantiGene^TM kits, comprising instructions and reagents such as amplification multimers, alkaline phosphatase labeled label probes, chemilumigenic substrate, capture probes immobilized on a solid support, and the like) are commercially available, e.g., from Panomics, Inc. (on the world wide web at (www.) panomics.com). Software for designing probe sets for a given mRNA target (i.e., for designing the regions of the CEs, LEs, and optionally BPs that are complementary to the target) is also commercially available (e.g., ProbeDesigner^TM from Panomics, Inc.; see also <nplcit id="ncit0017" npl-type="s"><text>Bushnell et al. (1999) "ProbeDesigner: for the design of probe sets for branched DNA (bDNA) signal amplification assays Bioinformatics 15:348-55</text></nplcit>).

0073The basic bDNA assay, however, permits detection of only a single target nucleic acid per assay, while, as described above, detection of multiple nucleic acids is frequently desirable.

0074Among other aspects, the present invention provides a multiplex bDNA assay that can be used for simultaneous detection of 10 or more target nucleic acids. The assay temperature and the stability of the complex between a single CE and its corresponding CP can be controlled such that binding of a single CE to a nucleic acid and to the CP is not sufficient to stably capture the nucleic acid on the surface to which the CP is bound, whereas simultaneous binding of two or more CEs to a nucleic acid can capture it on the surface. Requiring such cooperative hybridization of multiple CEs for capture of each nucleic acid of interest results in high specificity and low background from cross-hybridization of the CEs with other, non-target nucleic acids. For an assay to achieve high specificity and sensitivity, it preferably has a low background, resulting, e.g., from minimal cross-hybridization. Such low background and minimal cross-hybridization are typically substantially more difficult to achieve in a multiplex assay than a single-plex assay, because the number of potential nonspecific interactions are greatly increased in a multiplex assay due to the increased number of probes used in the assay (e.g., the greater number of CEs and LEs). Requiring multiple simultaneous CE-CP interactions for the capture of a target nucleic acid minimizes the chance that nonspecific capture will occur, even when some nonspecific CE-CP interactions do occur.

0075The methods of the invention can be used for multiplex detection of 10 or more nucleic acids simultaneously, for example, from even complex samples, without requiring prior purification of the nucleic acids, when the nucleic acids are present at low concentration, and/or in the presence of other, highly similar nucleic acids. In one aspect, the methods involve capture of the nucleic acids to particles (e.g., distinguishable subsets of microspheres), while in another aspect, the nucleic acids are captured to a spatially addressable solid support. Compositions and related to the methods are also provided.

<u>METHODS</u>

0076As noted, one aspect of the invention provides multiplex bDNA assays. Thus, one general class of embodiments includes methods of detecting 10 or more nucleic acids of interest. In the methods, a sample, a pooled population of particles, and 10 or more subsets of n capture extenders, wherein n is at least two, are provided. The sample comprises or is suspected of comprising the nucleic acids of interest. The pooled population of particles includes 10 or more subsets of particles, and a plurality of the particles in each subset are distinguishable from a plurality of the particles in every other subset. (Typically, substantially all of the particles in each subset are distinguishable from substantially all of the particles in every other subset.) The particles in each subset have associated therewith a different capture probe. Each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected subset of the particles. Each nucleic acid of interest can thus, by hybridizing to its corresponding subset of n capture extenders which are in turn hybridized to a corresponding capture probe, be associated with an identifiable subset of the particles.

0077The sample, the pooled population of particles, and the subsets of n capture extenders are contacted, any nucleic acid of interest present in the sample is hybridized to its corresponding subset of n capture extenders, and the subset of n capture extenders is hybridized to its corresponding capture probe. The hybridizing the nucleic acid of interest to the n capture extenders and the n capture extenders to the corresponding capture probe captures the nucleic acid on the subset of particles with which the capture extenders are associated. The hybridizing the subset of n capture extenders to the corresponding capture probe is performed at a hybridization temperature which is greater than a melting temperature T_m of a complex between each individual capture extender and its corresponding capture probe. Binding of a single capture extender to its corresponding nucleic acid (or to an extraneous nucleic acid) and capture probe is thus typically insufficient to capture the nucleic acid on the corresponding subset of particles. It will be evident that the hybridization temperature is typically less than a T_m of a complex between the nucleic acid of interest, all n corresponding capture extenders, and the corresponding capture probe.

0078To determine which subsets of particles have a nucleic acid of interest captured on the particles, one or more (e.g., two or more) label extenders and a label probe system comprising a label are hybridized to any nucleic acid of interest captured on the particles, and at least a portion of the particles from each subset are identified and the presence or absence of the label is detected on those particles. Since a correlation exists between a particular subset of particles and a particular nucleic acid of interest, which subsets of particles have the label present indicates which of the nucleic acids of interest were present in the sample.

0079The methods are useful for multiplex detection of nucleic acids, optionally highly multiplex detection. Thus, the two or more nucleic acids of interest (i.e., the nucleic acids to be detected) optionally comprise five or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or even 100 or more nucleic acids of interest. A like number of subsets of particles and subsets of CEs are provided; thus, the two or more subsets of particles can comprise five or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or even 100 or more subsets of particles, while the two or more subsets of n capture extenders can comprise five or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or even 100 or more subsets of n capture extenders.

0080Essentially any suitable particles, e.g., particles having distinguishable characteristics and to which capture probes can be attached, can be used. For example, in one preferred class of embodiments, the particles are microspheres. The microspheres of each subset can be distinguishable from those of the other subsets, e.g., on the basis of their fluorescent emission spectrum, their diameter, or a combination thereof. For example, the microspheres of each subset can be labeled with a unique fluorescent dye or mixture of such dyes, quantum dots with distinguishable emission spectra, and/or the like. As another example, the particles of each subset can be identified by an optical barcode, unique to that subset, present on the particles.

0081The particles optionally have additional desirable characteristics. For example, the particles can be magnetic or paramagnetic, which provides a convenient means for separating the particles from solution, e.g., to simplify separation of the particles from any materials not bound to the particles.

0082As noted, each of the 10 or more subsets of capture extenders includes n capture extenders, where n is at least two. Preferably, n is at least three, and n can be at least four or at least five or more. Typically, but not necessarily, n is at most ten. For example, n can be between three and ten, e.g., between five and ten or between five and seven, inclusive. Use of fewer capture extenders can be advantageous, for example, in embodiments in which nucleic acids of interest are to be specifically detected from samples including other nucleic acids with sequences very similar to that of the nucleic acids of interest. In other embodiments (e.g., embodiments in which capture of as much of the nucleic acid as possible is desired), however, n can be more than 10, e.g., between 20 and 50. n can be the same for all of the subsets of capture extenders, but it need not be; for example, one subset can include three capture extenders while another subset includes five capture extenders. The n capture extenders in a subset preferably hybridize to nonoverlapping polynucleotide sequences in the corresponding nucleic acid of interest. The nonoverlapping polynucleotide sequences can, but need not be, consecutive within the nucleic acid of interest.

0083Each capture extender is capable of hybridizing to its corresponding capture probe. The capture extender typically includes a polynucleotide sequence C-1 that is complementary to a polynucleotide sequence C-2 in its corresponding capture probe. In one aspect, C-1 and C-2 are 20 nucleotides or less in length. In one class of embodiments, C-1 and C-2 are between 9 and 17 nucleotides in length (inclusive), preferably between 12 and 15 nucleotides (inclusive). For example, C-1 and C-2 can be 14, 15, 16, or 17 nucleotides in length, or they can be between 9 and 13 nucleotides in length (e.g., for lower hybridization temperatures, e.g., hybridization at room temperature).

0084The capture probe can include polynucleotide sequence in addition to C-2, or C-2 can comprise the entire polynucleotide sequence of the capture probe. For example, each capture probe optionally includes a linker sequence between the site of attachment of the capture probe to the particles and sequence C-2 (e.g., a linker sequence containing 8 Ts, as just one possible example).

0085It will be evident that the amount of overlap between each individual capture extender and its corresponding capture probe (i.e., the length of C-1 and C-2) affects the T_m of the complex between that capture extender and capture probe, as does, e.g., the GC base content of sequences C-1 and C-2. Typically, all the capture probes are the same length (as are sequences C-1 and C-2) from subset of particles to subset. However, depending, e.g., on the precise nucleotide sequence of C-2, different capture probes optionally have different lengths and/or different length sequences C-2, to achieve the desired T_m. Different capture probe-capture extender complexes optionally have the same or different T_ms.

0086It will also be evident that the number of capture extenders required for stable capture of a nucleic acid depends, in part, on the amount of overlap between the capture extenders and the capture probe (i.e., the length of C-1 and C-2). For example, if n is 5-7 for a 14 nucleotide overlap, n could be 3-5 for a 15 nucleotide overlap or 2-3 for a 16 nucleotide overlap.

0087As noted, the hybridizing the subset of n capture extenders to the corresponding capture probe is performed at a hybridization temperature which is greater than a melting temperature T_m of a complex between each individual capture extender and its corresponding capture probe. The hybridization temperature is typically about 5°C or more greater than the T_m, e.g., about 7°C or more, about 10°C or more, about 12°C or more, about 15°C or more, about 17°C or more, or even about 20°C or more greater than the T_m.

0088Stable capture of nucleic acids of interest, e.g., while minimizing capture of extraneous nucleic acids (e.g., those to which n-1 or fewer of the capture extenders bind) can be achieved, for example, by balancing n (the number of capture extenders), the amount of overlap between the capture extenders and the capture probe (the length of C-1 and C-2), and/or the stringency of the conditions under which the capture extenders, the nucleic acids, and the capture probes are hybridized.

0089Appropriate combinations of n, amount of complementarity between the capture extenders and the capture probe, and stringency of hybridization can, for example, be determine experimentally by one of skill in the art. For example, as illustrated in the experiments described in Example 1 herein, a particular value of n and a particular set of hybridization conditions can be selected, while the number of nucleotides of complementarity between the capture extenders and the capture probe is varied until hybridization of the n capture extenders to a nucleic acid captures the nucleic acid while hybridization of a single capture extender does not efficiently capture the nucleic acid. The experiments described in Example 1 also illustrate selection of n, amount of complementarity, and stringency of hybridization such that the desired nucleic acid of interest is captured while other nucleic acids present in the sample are not efficiently captured. Stringency can be controlled, for example, by controlling the formamide concentration, chaotropic salt concentration, salt concentration, pH, organic solvent content, and/or hybridization temperature.

0090As noted, the T_m of any nucleic acid duplex can be directly measured, using techniques well known in the art. For example, a thermal denaturation curve can be obtained for the duplex, the midpoint of which corresponds to the T_m. It will be evident that such denaturation curves can be obtained under conditions having essentially any relevant pH, salt concentration, solvent content, and/or the like.

0091The T_m for a particular duplex (e.g., an approximate T_m) can also be calculated. For example, the T_m for an oligonucleotide-target duplex can be estimated using the following algorithm, which incorporates nearest neighbor thermodynamic parameters: Tm (Kelvin) = ΔH°/(ΔS° + R lnC_t), where the changes in standard enthalpy (ΔH°) and entropy (ΔS°) are calculated from nearest neighbor thermodynamic parameters (see, e.g., <nplcit id="ncit0018" npl-type="s"><text>SantaLucia (1998) "A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics" Proc. Natl. Acad. Sci. USA 95:1460-1465</text></nplcit>, <nplcit id="ncit0019" npl-type="s"><text>Sugimoto et al. (1996) "Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes" Nucleic Acids Research 24: 4501-4505</text></nplcit>, <nplcit id="ncit0020" npl-type="s"><text>Sugimoto et al. (1995) "Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes" Biochemistry 34:11211-11216</text></nplcit>, and et al. (<nplcit id="ncit0021" npl-type="s"><text>1998) "Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs" Biochemistry 37: 14719-14735</text></nplcit>), R is the ideal gas constant (1.987 cal·K^-1mole^-1), and C_t is the molar concentration of the oligonucleotide. The calculated T_m is optionally corrected for salt concentration, e.g., Na⁺ concentration, using the formula 1/T_m(Na⁺) = 1/T_m(1M) + (4.29<i>f</i>(G·C)-3.95)x10^-5 ln[Na⁺] + 9.40x10^-6 ln²[Na⁺]. See, e.g., <nplcit id="ncit0022" npl-type="s"><text>Owczarzy et al. (2004) "Effects of Sodium Ions on DNA Duplex Oligomers: Improved Predictions of Melting Temperatures" Biochemistry 43:3537-3554</text></nplcit> for further details. A Web calculator for estimating Tm using the above algorithms is available on the Internet at scitools.idtdna.com/analyzer/oligocalc.asp. Other algorithms for calculating T_m are known in the art and are optionally applied to the present invention.

0092In one class of embodiments, contacting the sample, the pooled population of particles, and the subsets of n capture extenders comprises combining the sample with the subsets of n capture extenders to form a mixture, and then combining the mixture with the pooled population of particles. In this class of embodiments, the capture extenders typically hybridize first to the corresponding nucleic acid of interest and then to the corresponding particle-associated capture probe. The hybridizations can, however, occur simultaneously or even in the opposite order. Thus, in another exemplary class of embodiments, contacting the sample, the pooled population of particles, and the subsets of n capture extenders comprises combining the sample, the subsets of capture extenders, and the pooled population of particles.

0093In a preferred class of embodiments, hybridizing one or more label extenders and a label probe system to any nucleic acid of interest captured on the particles comprises providing two or more subsets of one or more label extenders, wherein each subset of label extenders is capable of hybridizing to one of the nucleic acids of interest, hybridizing any nucleic acid of interest captured on the particles to its corresponding subset of label extenders, and hybridizing the label probe system to the label extenders. The two or more subsets of label extenders can include, e.g., five or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or even 100 or more subsets of label extenders. Typically, each subset includes two or more label extenders. The hybridizations can be performed simultaneously or sequentially, in essentially any order. As just one example, the capture extenders and label extenders can be hybridized to their corresponding nucleic acids of interest, then the capture extenders can be hybridized to their corresponding capture probes, and then the label probe system can be hybridized to the label extenders.

0094The label probe system optionally includes an amplification multimer and a plurality of label probes, wherein the amplification multimer is capable of hybridizing to a label extender and to a plurality of label probes. In another aspect, the label probe system includes a preamplifier, a plurality of amplification multimers, and a plurality of label probes, wherein the preamplifier hybridizes to the label extenders, and the amplification multimers hybridize to the preamplifier and to the plurality of label probes. As another example, the label probe system can include only label probes, which hybridize directly to the label extenders. In one class of embodiments, the label probe comprises the label. In other embodiments, the label probe is configured to bind a label; for example, a biotinylated label probe can bind to a streptavidin-associated label.

0095The label can be essentially any convenient label that directly or indirectly provides a detectable signal. In one aspect, the label is a fluorescent label (e.g., a fluorophore or quantum dot). Detecting the presence of the label on the particles thus comprises detecting a fluorescent signal from the label. Fluorescent emission by the label is typically distinguishable from any fluorescent emission by the particles, e.g., microspheres, and many suitable fluorescent label-fluorescent microsphere combinations are possible. As other examples, the label can be a luminescent label, a light-scattering label (e.g., colloidal gold particles), or an enzyme (e.g., HRP).

0096The methods can optionally be used to quantitate the amounts of the nucleic acids of interest present in the sample. For example, in one class of embodiments, an intensity of a signal from the label is measured, e.g., for each subset of particles, and correlated with a quantity of the corresponding nucleic acid of interest present.

0097As noted, blocking probes are optionally also hybridized to the nucleic acids of interest, which can reduce background in the assay. For a given nucleic acid of interest, the corresponding capture extenders, label extenders, and optional blocking probes are preferably complementary to physically distinct, nonoverlapping sequences in the nucleic acid of interest, which are preferably, but not necessarily, contiguous. The T_ms of the capture extender-nucleic acid, label extender-nucleic acid, and blocking probe-nucleic acid complexes are preferably greater than the hybridization temperature, e.g., by 5°C or 10°C or preferably by 15°C or more, such that these complexes are stable at the hybridization temperature. Potential CE and LE sequences (e.g., potential sequences C-3. and L-1) are optionally examined for possible interactions with non-corresponding nucleic acids of interest, LEs or CEs, the amplification multimer, the preamplifier, the label probe, and/or any relevant genomic sequences, for example; sequences expected to cross-hybridize with undesired nucleic acids are typically not selected for use in the CEs or LEs. See, e.g., Example 1 herein and <nplcit id="ncit0023" npl-type="s"><text>Player et al. (2001) " Single-copy gene detection using branched DNA (bDNA) in situ hybridization" J Histochem Cytochem 49:603-611</text></nplcit>. Examination can be, e.g., visual (e.g., visual examination for complementarity), computational (e.g., computation and comparison of binding free energies), and/or experimental (e.g., cross-hybridization experiments). Capture probe sequences are preferably similarly examined, to ensure that the polynucleotide sequence C-1 complementary to a particular capture probe's sequence C-2 is not expected to cross-hybridize with any of the other capture probes that are to be associated with other subsets of particles. See, e.g., Example 1 herein.

0098At any of various steps, materials not captured on the particles are optionally separated from the particles. For example, after the capture extenders, nucleic acids, label extenders, blocking probes, and particle-bound capture probes are hybridized, the particles are optionally washed to remove unbound nucleic acids and probes; after the label extenders and amplification multimer are hybridized, the particles are optionally washed to remove unbound amplification multimer; and/or after the label probes are hybridized to the amplification multimer, the particles are optionally washed to remove unbound label probe prior to detection of the label.

0099One or more of the subsets of particles is optionally isolated, whereby the associated nucleic acid of interest is isolated. The isolated nucleic acid can optionally be removed from the particles and/or subjected to further manipulation, if desired (e.g., amplification by PCR or the like).

0100An exemplary embodiment is schematically illustrated in <figref idref="f0002 f0003"><b>Figure 2</b></figref>. <b>Panel A</b> illustrates three distinguishable subsets of microspheres <b>201</b>, <b>202</b>, and <b>203</b>, which have associated therewith capture probes <b>204</b>, <b>205</b>, and <b>206</b>, respectively. Each capture probe includes a sequence C-2 (<b>250</b>), which is different from subset to subset of microspheres. The three subsets of microspheres are combined to form pooled population <b>208</b> (<b>Panel B</b>). A subset of three capture extenders is provided for each nucleic acid of interest; subset <b>211</b> for nucleic acid <b>214</b>, subset <b>212</b> for nucleic acid <b>215</b> which is not present, and subset <b>213</b> for nucleic acid <b>216</b>. Each capture extender includes sequences C-1 (<b>251</b>, complementary to the respective capture probe's sequence C-2) and C-3 (<b>252</b>, complementary to a sequence in the corresponding nucleic acid of interest). Three subsets of label extenders (<b>221</b>, <b>222</b>, and <b>223</b> for nucleic acids <b>214</b>, <b>215</b>, and <b>216</b>, respectively) and three subsets of blocking probes (<b>224</b>, <b>225</b>, and <b>226</b> for nucleic acids <b>214</b>, <b>215</b>, and <b>216</b>, respectively) are also provided. Each label extender includes sequences L-1 (<b>254</b>, complementary to a sequence in the corresponding nucleic acid of interest) and L-2 (<b>255</b>, complementary to M-1). Non-target nucleic acids <b>230</b> are also present in the sample of nucleic acids.

0101Nucleic acids <b>214</b> and <b>216</b> are hybridized to their corresponding subset of capture extenders (<b>211</b> and <b>213</b>, respectively), and the capture extenders are hybridized to the corresponding capture probes (<b>204</b> and <b>206</b>, respectively), capturing nucleic acids <b>214</b> and <b>216</b> on microspheres <b>201</b> and <b>203</b>, respectively (<b>Panel C</b>). Materials not bound to the microspheres (e.g., capture extenders <b>212</b>, nucleic acids <b>230</b>, etc.) are separated from the microspheres by washing. Label probe system <b>240</b> including amplification multimer <b>241</b> (which includes sequences M-1 <b>257</b> and M-2 <b>258</b>) and label probe <b>242</b> (which contains label <b>243</b>) is hybridized to label extenders <b>221</b> and <b>223</b>, which are hybridized to nucleic acids <b>214</b> and <b>216</b>, respectively (<b>Panel D</b>). Materials not captured on the microspheres are optionally removed by washing the microspheres. Microspheres from each subset are identified, e.g., by their fluorescent emission spectrum (λ₂ and λ₃, <b>Panel E</b>), and the presence or absence of the label on each subset of microspheres is detected (λ₁, <b>Panel E</b>). Since each nucleic acid of interest is associated with a distinct subset of microspheres, the presence of the label on a given subset of microspheres correlates with the presence of the corresponding nucleic acid in the original sample.

0102As depicted in <figref idref="f0002 f0003"><b>Figure 2</b></figref>, all of the label extenders in all of the subsets typically include an identical sequence L-2. Optionally, however, different label extenders (e.g., label extenders in different subsets) can include different sequences L-2. Also as depicted in <figref idref="f0002 f0003"><b>Figure 2</b></figref>, each capture probe typically includes a single sequence C-2 and thus hybridizes to a single capture extender. Optionally, however, a capture probe can include two or more sequences C-2 and hybridize to two or more capture extenders. Similarly, as depicted, each of the capture extenders in a particular subset typically includes an identical sequence C-1, and thus only a single capture probe is needed for each subset of particles; however, different capture extenders within a subset optionally include different sequences C-1 (and thus hybridize to different sequences C-2, within a single capture probe or different capture probes on the surface of the corresponding subset of particles).

0103The methods can be used to detect the presence of the nucleic acids of interest in essentially any type of sample. For example, the sample can be derived from an animal, a human, a plant, a cultured cell, a virus, a bacterium, a pathogen, and/or a microorganism. The sample optionally includes a cell lysate, an intercellular fluid, a bodily fluid (including, but not limited to, blood, serum, saliva, urine, sputum, or spinal fluid), and/or a conditioned culture medium, and is optionally derived from a tissue (e.g., a tissue homogenate), a biopsy, and/or a tumor. Similarly, the nucleic acids can be essentially any desired nucleic acids. As just a few examples, the nucleic acids of interest can be derived from one or more of an animal, a human, a plant, a cultured cell, a microorganism, a virus, a bacterium, or a pathogen.

0104As noted, the methods can be used for gene expression analysis. Accordingly, in one class of embodiments, the two or more nucleic acids of interest comprise two or more mRNAs. The methods can also be used for clinical diagnosis and/or detection of microorganisms, e.g., pathogens. Thus, in certain embodiments, the nucleic acids include bacterial and/or viral genomic RNA and/or DNA (double-stranded or single-stranded), plasmid or other extra-genomic DNA, or other nucleic acids derived from microorganisms (pathogenic or otherwise). It will be evident that double-stranded nucleic acids of interest will typically be denatured before hybridization with capture extenders, label extenders, and the like.

0105Due to cooperative hybridization of multiple capture extenders to a nucleic acid of interest, for example, even nucleic acids present at low concentration can be captured and detected. Thus, in one class of embodiments, at least one of the nucleic acids of interest is present in the sample in a non-zero amount of 200 amol or less, 150 amol or less, 100 amol or less, 50 amol or less, 10 amol or less, 1 amol or less, or even 0.1 amol or less, 0.01 amol or less, 0.001 amol or less, or 0.0001 amol or less. Similarly, two nucleic acids of interest can be captured and detected simultaneously, even when they differ in concentration by 1000-fold or more in the sample. The methods are thus extremely versatile.

0106Capture of a particular nucleic acid is optionally quantitative. Thus, in one exemplary class of embodiments, the sample includes a first nucleic acid of interest, and at least 30%, at least 50%, at least 80%, at least 90%, at least 95%, or even at least 99% of a total amount of the first nucleic acid present in the sample is captured on a first subset of particles. Second, third, etc. nucleic acids can similarly be quantitatively captured. Such quantitative capture can occur without capture of a significant amount of undesired nucleic acids, even those of very similar sequence to the nucleic acid of interest.

0107Thus, in one class of embodiments, the sample comprises or is suspected of comprising a first nucleic acid of interest and a second nucleic acid which has a polynucleotide sequence which is 95% or more identical to that of the first nucleic acid (e.g., 96% or more, 97% or more, 98% or more, or even 99% or more identical). The first nucleic acid, if present in the sample, is captured on a first subset of particles, while the second nucleic acid comprises 1% or less of a total amount of nucleic acid captured on the first subset of particles (e.g., 0.5% or less, 0.2% or less, or even 0.1% or less). The second nucleic acid can be another nucleic acid of interest or simply any nucleic acid. Typically, capture extenders are chosen that hybridize to regions of the first nucleic acid having the greatest sequence difference from the second nucleic acid.

0108As just one example of how closely related nucleic acids can be distinguished using the methods of the invention, different splice variants of a given mRNA can be distinguished. Thus, in one class of embodiments, the sample comprises a first nucleic acid of interest and a second nucleic acid, where the first nucleic acid is a first splice variant and the second nucleic acid is a second splice variant of the given mRNA. A first subset of n capture extenders is capable of hybridizing to the first splice variant, of which at most n-1 capture extenders are capable of hybridizing to the second splice variant. Optionally, at least 80% or more, 90% or more, or 95% or more of the first splice variant is captured on a first subset of particles while at most 10% or less, 5% or less, 3% or less, or 1% or less of the second splice variant is captured on the first subset of particles. Preferably, hybridization of the n capture extenders to the first splice variant captures the first splice variant on a first subset of particles while hybridization of the at most n-1 capture extenders to the second splice variant does not capture the second splice variant on the first subset of particles.

0109An exemplary embodiment illustrating detection of two splice variants is schematically depicted in <figref idref="f0004"><b>Figure 3</b></figref><b>.</b> In this example, three CEs <b>11</b> hybridize to first splice variant <b>21,</b> one to each exon <b>(24</b> and <b>26)</b> and one to splice junction <b>27</b> (the only sequence found in first splice variant <b>21</b> and not also found in second splice variant <b>22);</b> two of these bind to second splice variant <b>22.</b> Similarly, three CEs <b>12</b> bind to second splice variant <b>22,</b> one to intron <b>25</b> and one to each of the splice junctions; none of these bind to first splice variant <b>21.</b> Through cooperative hybridization of the CEs to the splice variants and to the corresponding capture probes <b>(4</b> and <b>5),</b> splice variants <b>21</b> and <b>22</b> are each captured specifically only on the corresponding subset of microspheres (<b>1</b> and <b>2,</b> respectively). LEs <b>14</b> and <b>15</b> are hybridized with splice variants <b>21</b> and <b>22,</b> respectively, and BPs <b>13</b> are hybridized with first splice variant <b>21.</b> Amplification multimers <b>30</b> are then hybridized to the LEs, and label probes <b>31</b> are hybridized to the amplification multimers. Microspheres from the two subsets are then identified, and the presence or absence of the label on the microspheres is detected and correlated with the presence or absence of the corresponding splice variant. For additional details on detection of splice variants, see <patcit id="pcit0030" dnum="US50159803P" dnum-type="L"><text>U.S. patent application 60/501,598, filed September 9, 2003, by Yuling Luo</text></patcit>, entitled "Methods of capturing, detecting, and quantitating splice variants." Optionally, for any nucleic acid, hybridization of a first subset of n capture extenders to a first nucleic acid captures the first nucleic acid on a first subset of particles while hybridization of at most n-1 of the capture extenders to a second nucleic acid does not capture the second nucleic acid on the first subset of particles.

0110It will be evident that nucleic acids that do not have 100% identical sequences are alternatively optionally captured on the same subset of particles, if desired. For example, a first and a second nucleic acid are optionally both captured on a first subset of particles, through binding of the same or different subsets of capture extenders. The first and second nucleic acids can be closely related; for example, splice variants of a particular mRNA, different alleles of a gene, somatic mutations, homologs, or the like.

0111A capture probe and/or capture extender optionally comprises at least one non-natural nucleotide. For example, a capture probe and the corresponding capture extender optionally comprise, at complementary positions, at least one pair of non-natural nucleotides that base pair with each other but that do not Watson-Crick base pair with the bases typical to biological DNA or RNA (i.e., A, C, G, T, or U). Examples of nonnatural nucleotides include, but are not limited to, Locked NucleicAcid^™ nucleotides (available from Exiqon A/S, (www.) exiqon.com; see, e.g., <nplcit id="ncit0024" npl-type="s"><text>SantaLucia Jr. (1998) Proc Natl Acad Sci 95:1460-1465</text></nplcit>) and isoG, isoC, and other nucleotides used in the AEGIS system (Artificially Expanded Genetic Information System, available from EraGen Biosciences, (www.) eragen.com; see, e.g., <patcit id="pcit0031" dnum="USPN6001983A"><text>USPN 6,001,983</text></patcit>, <patcit id="pcit0032" dnum="USPN6037120A"><text>USPN 6,037,120</text></patcit>, and <patcit id="pcit0033" dnum="USPN6140496A"><text>USPN 6,140,496</text></patcit>). Use of such non-natural base pairs (e.g., isoG-isoC base pairs) in the capture probes and capture extenders can, for example, reduce background and/or simplify probe design by decreasing cross hybridization, or it can permit use of shorter CPs and CEs when the non-natural base pairs have higher binding affinities than do natural base pairs. (Non-natural nucleotides can similarly be included in the label extenders, preamplifiers, amplification multimers, and/or label probes, if desired.)

0112The preceding embodiments include capture of the nucleic acids of interest on particles. Alternatively, the nucleic acids can be captured at different positions on a non-particulate, spatially addressable solid support. Accordingly, another general class of embodiments includes methods of detecting 10 or more nucleic acids of interest. In the methods, a sample, a solid support, and 10 or more subsets of n capture extenders, wherein n is at least two, are provided. The sample comprises or is suspected of comprising the nucleic acids of interest. The solid support comprises 10 or more capture probes, each of which is provided at a selected position on the solid support. Each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected position on the solid support. Each nucleic acid of interest can thus, by hybridizing to its corresponding subset of n capture extenders which are in turn hybridized to a corresponding capture probe, be associated with, e.g., a known, predetermined location on the solid support. The sample, the solid support, and the subsets of n capture extenders are contacted, any nucleic acid of interest present in the sample is hybridized to its corresponding subset of n capture extenders, and the subset of n capture extenders is hybridized to its corresponding capture probe. The hybridizing the nucleic acid of interest to the n capture extenders and the n capture extenders to the corresponding capture probe captures the nucleic acid on the solid support at the selected position with which the capture extenders are associated. To determine which positions on the solid support have a nucleic acid of interest captured at that position, one or more (e.g., two or more) label extenders and a label probe system comprising a label are hybridized to any nucleic acid of interest captured on the solid support, and the presence or absence of the label at the selected positions on the solid support is detected. Since a correlation exists between a particular position on the support and a particular nucleic acid of interest, which positions have a label present indicates which of the nucleic acids of interest were present in the sample.

0113The hybridizing the subset of n capture extenders to the corresponding capture probe is typically performed at a hybridization temperature which is greater than a melting temperature T_m of a complex between each individual capture extender and its corresponding capture probe. For example, the hybridization temperature can be about 5°C or more greater than the T_m, e.g., about 7°C or more, about 10°C or more, about 12°C or more, about 15°C or more, about 17°C or more, or even about 20°C or more greater than the T_m.

0114The methods are useful for multiplex detection of nucleic acids, optionally highly multiplex detection. Thus, the two or more nucleic acids of interest (i.e., the nucleic acids to be detected) optionally comprise five or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 100 or more, 10³ or more, or 10⁴ or more nucleic acids of interest. A like number of selected positions on the solid support and subsets of CEs are provided; thus, the two or more selected positions can comprise five or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 100 or more, 10³ or more, or 10⁴ or more selected positions, while the two or more subsets of n capture extenders can comprise five or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 100 or more, 10³ or more, or 10⁴ or more subsets of n capture extenders.

0115The solid support typically has a planar surface and is typically rigid, but essentially any spatially addressable solid support can be adapted to the practice of the present invention. Exemplary materials for the solid support include, but are not limited to, glass, silicon, silica, quartz, plastic, polystyrene, nylon, and nitrocellulose. As just one examples, an array of capture probes can be formed at selected positions on a glass slide as the solid support.

0116Essentially all of the features noted for the methods above apply to these embodiments as well, as relevant; for example, with respect to number of capture extenders per subset, composition of the label probe system, type of label, use of blocking probes, configuration of the capture extenders, capture probes, label extenders, and/or blocking probes, quantitation of the nucleic acids of interest, source of the sample and/or nucleic acids, and/or the like.

0117For example, in one class of embodiments, contacting the sample, the solid support, and the subsets of n capture extenders comprises combining the sample with the subsets of n capture extenders to form a mixture, and then contacting the mixture with the solid support. In this class of embodiments, the capture extenders typically hybridize first to the corresponding nucleic acid of interest and then to the corresponding particle-associated capture probe. In other embodiments, however, the hybridizations can occur simultaneously or even in the opposite order.

0118At any of various steps, materials not captured on the solid support are optionally separated from the solid support. For example, after the capture extenders, nucleic acids, label extenders, blocking probes, and support-bound capture probes are hybridized, the solid support is optionally washed to remove unbound nucleic acids and probes; after the label extenders and amplification multimer are hybridized, the support is optionally washed to remove unbound amplification multimer; and/or after the label probes are hybridized to the amplification multimer, the support is optionally washed to remove unbound label probe prior to detection of the label.

0119As for the embodiments described above, capture of a particular nucleic acid is optionally quantitative. Thus, in one exemplary class of embodiments, the sample includes a first nucleic acid of interest, and at least 30%, at least 50%, at least 80%, at least 90%, at least 95%, or even at least 99% of a total amount of the first nucleic acid present in the sample is captured at a first selected position on the solid support. Second, third, etc. nucleic acids can similarly be quantitatively captured. Such quantitative capture can occur without capture of a significant amount of undesired nucleic acids, even those of very similar sequence to the nucleic acid of interest.

0120Thus, in one class of embodiments, the sample comprises or is suspected of comprising a first nucleic acid of interest and a second nucleic acid which has a polynucleotide sequence which is 95% or more identical to that of the first nucleic acid (e.g., 96% or more, 97% or more, 98% or more, or even 99% or more identical). The first nucleic acid, if present in the sample, is captured at a first selected position on the solid support, while the second nucleic acid comprises 1% or less of a total amount of nucleic acid captured at the first position (e.g., 0.5% or less, 0.2% or less, or even 0.1% or less). The second nucleic acid can be another nucleic acid of interest or simply any nucleic acid. Typically, capture extenders are chosen that hybridize to regions of the first nucleic acid having the greatest sequence difference from the second nucleic acid.

0121As just one example of how closely related nucleic acids can be distinguished using the methods of the invention, different splice variants of a given mRNA can be distinguished. Thus, in one class of embodiments, the sample comprises a first nucleic acid of interest and a second nucleic acid, where the first nucleic acid is a first splice variant and the second nucleic acid is a second splice variant of the given mRNA. A first subset of n capture extenders is capable of hybridizing to the first splice variant, of which at most n-1 capture extenders are capable of hybridizing to the second splice variant. Optionally, at least 80% or more, 90% or more, or 95% or more of the first splice variant is captured at a first selected position on the solid support while at most 10% or less, 5% or less, 3% or less, or 1% or less of the second splice variant is captured at the first position. Preferably, hybridization of the n capture extenders to the first splice variant captures the first splice variant at a first selected position on the solid support while hybridization of the at most n-1 capture extenders to the second splice variant does not capture the second splice variant at the first position.

0122It will be evident that nucleic acids that do not have 100% identical sequences are alternatively optionally captured at the same position of the support, if desired. For example, a first and a second nucleic acid are optionally both captured at a first position, through binding of the same or different subsets of capture extenders. The first and second nucleic acids can be closely related; for example, splice variants of a particular mRNA, different alleles of a gene, somatic mutations, homologs, or the like.

0123An exemplary embodiment is schematically illustrated in <figref idref="f0005 f0006"><b>Figure 4</b></figref><b>. Panel A</b> depicts solid support <b>301</b> having nine capture probes provided on it at nine selected positions (e.g., <b>334-336</b>). <b>Panel B</b> depicts a cross section of solid support <b>301,</b> with distinct capture probes <b>304, 305,</b> and <b>306</b> at different selected positions on the support (<b>334, 335,</b> and <b>336,</b> respectively). A subset of capture extenders is provided for each nucleic acid of interest. Only three subsets are depicted; subset <b>311</b> for nucleic acid <b>314,</b> subset <b>312</b> for nucleic acid <b>315</b> which is not present, and subset <b>313</b> for nucleic acid <b>316.</b> Each capture extender includes sequences C-1 (<b>351,</b> complementary to the respective capture probe's sequence C-2) and C-3 (<b>352,</b> complementary to a sequence in the corresponding nucleic acid of interest). Three subsets of label extenders (<b>321, 322,</b> and <b>323</b> for nucleic acids <b>314, 315,</b> and <b>316,</b> respectively) and three subsets of blocking probes (<b>324, 325,</b> and <b>326</b> for nucleic acids <b>314, 315,</b> and <b>316,</b> respectively) are also depicted (although nine would typically be provided, one for each nucleic acid of interest). Each label extender includes sequences <b>L-1</b> (<b>354,</b> complementary to a sequence in the corresponding nucleic acid of interest) and <b>L-2</b> (<b>355,</b> complementary to <b>M-1</b>). Non-target nucleic acids <b>330</b> are also present in the sample of nucleic acids.

0124Nucleic acids <b>314</b> and <b>316</b> are hybridized to their corresponding subset of capture extenders (<b>311</b> and <b>313,</b> respectively), and the capture extenders are hybridized to the corresponding capture probes (<b>304</b> and <b>306,</b> respectively), capturing nucleic acids <b>314</b> and <b>316</b> at selected positions <b>334</b> and <b>336,</b> respectively (<b>Panel C</b>). Materials not bound to the solid support (e.g., capture extenders <b>312,</b> nucleic acids <b>330,</b> etc.) are separated from the support by washing. Label probe system <b>340</b> including amplification multimer <b>341</b> (which includes sequences <b>M-1 357</b> and <b>M-2 358</b>) and label probe <b>342</b> (which contains label <b>343</b>) is hybridized to label extenders <b>321</b> and <b>323,</b> which are hybridized to nucleic acids <b>314</b> and <b>316,</b> respectively (<b>Panel D</b>)<b>.</b> Materials not captured on the solid support are optionally removed by washing the support, and the presence or absence of the label at each position on the solid support is detected. Since each nucleic acid of interest is associated with a distinct position on the support, the presence of the label at a given position on the support correlates with the presence of the corresponding nucleic acid in the original sample.

0125The methods of the present invention offer a number of advantages. For example, a single array of capture probes at selected positions on a solid support can be manufactured, and this single array can be used to detect essentially any desired group of nucleic acids of interest simply by synthesizing appropriate subsets of capture extenders, label extenders, and the like. A new array need not be manufactured for each new group of nucleic acids to be detected, unlike conventional microarray technologies in which arrays of target-specific probes attached to a solid support are utilized, necessitating the manufacture of a new array for each new group of target nucleic acids to be detected. Similarly, a single population of subsets of particles comprising capture probes can be manufactured and used for detection of essentially any desired group of nucleic acids of interest.

COMPOSITIONS

0126Compositions related to the methods are another feature of the invention. Thus, one general class of embodiments provides a composition for detecting 10 or more nucleic acids of interest, the composition that includes or more subsets of particles, 10 or more subsets of n capture extenders, wherein n is at least two, 10 or more subsets of one or more label extenders, and a label probe system comprising a label. A plurality of the particles in each subset are distinguishable from a plurality of the particles in every other subset. (Typically, substantially all of the particles in each subset are distinguishable from substantially all of the particles in every other subset.) The particles in each subset have associated therewith a different capture probe. Each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected subset of the particles. When the nucleic acid of interest corresponding to a subset of n capture extenders is present in the composition and is hybridized to the subset of n capture extenders, which are hybridized to the corresponding capture probe, the nucleic acid of interest is hybridized to the subset of n capture extenders at a hybridization temperature which is greater than a melting temperature T_m of a complex between each individual capture extender and the capture probe.

0127The composition optionally includes a sample comprising or suspected of comprising at least one of the nucleic acids of interest, e.g., two or more, three or more, etc. nucleic acids. In one class of embodiments, the composition comprises one or more of the nucleic acids of interest. Each nucleic acid of interest is hybridized to its corresponding subset of n capture extenders, and the corresponding subset of n capture extenders is hybridized to its corresponding capture probe. Each nucleic acid of interest is thus associated with an identifiable subset of the particles. The composition is maintained at the hybridization temperature.

0128As noted, the hybridization temperature is greater than the T_m of each of the individual CE-CP complexes. The hybridization temperature is typically about 5°C or more greater than the T_m, e.g., about 7°C or more, about 10°C or more, about 12°C or more, about 15°C or more, about 17°C or more, or even about 20°C or more greater than the T_m.

0129Essentially all of the features noted for the methods above apply to these embodiments as well, as relevant; for example, with respect to number of capture extenders per subset, composition of the label probe system, type of label, inclusion of blocking probes, configuration of the capture extenders, capture probes, label extenders, and/or blocking probes, number of nucleic acids of interest and of subsets of particles, capture extenders and label extenders, type of particles, source of the sample and/or nucleic acids, and/or the like.

0130As noted, even nucleic acids present at low concentration can be captured. Thus, in one class of embodiments, at least one of the nucleic acids of interest is present in the composition in a non-zero amount of 200 amol or less, 150 amol or less, 100 amol or less, 50 amol or less, 10 amol or less, 1 amol or less, or even 0.1 amol or less, 0.01 amol or less, 0.001 amol or less, or 0.0001 amol or less. Similarly, two nucleic acids of interest can be captured simultaneously, even when they differ in concentration by 1000-fold or more in the composition.

0131Capture of a particular nucleic acid on the particles is optionally quantitative. Thus, in one exemplary class of embodiments, the composition includes a first nucleic acid of interest, and at least 30%, at least 50%, at least 80%, at least 90%, at least 95%, or even at least 99% of a total amount of the first nucleic acid present in the composition is captured on a first subset of particles. Second, third, etc. nucleic acids can similarly be quantitatively captured. Such quantitative capture can occur without capture of a significant amount of undesired nucleic acids, even those of very similar sequence to the nucleic acid of interest.

0132Thus, in one class of embodiments, the composition comprises or is suspected of comprising a first nucleic acid of interest and a second nucleic acid which has a polynucleotide sequence which is 95% or more identical to that of the first nucleic acid (e.g., 96% or more, 97% or more, 98% or more, or even 99% or more identical). The first nucleic acid, if present in the composition, is captured on a first subset of particles, while the second nucleic acid comprises 1% or less of a total amount of nucleic acid captured on the first subset of particles (e.g., 0.5% or less, 0.2% or less, or even 0.1% or less). The second nucleic acid can be another nucleic acid of interest or simply any nucleic acid. Typically, capture extenders are chosen that hybridize to regions of the first nucleic acid having the greatest sequence difference from the second nucleic acid.

0133In one exemplary class of embodiments in which related nucleic acids are differentially captured, the composition comprises a first nucleic acid of interest and a second nucleic acid, where the first nucleic acid is a first splice variant and the second nucleic acid is a second splice variant of a given mRNA. A first subset of n capture extenders is capable of hybridizing to the first splice variant, of which at most n-1 capture extenders are capable of hybridizing to the second splice variant. Optionally, at least 80% or more, 90% or more, or 95% or more of the first splice variant is captured on a first subset of particles while at most 10% or less, 5% or less, 3% or less, or 1% or less of the second splice variant is captured on the first subset of particles. Preferably, a first subset of n capture extenders is hybridized to the first splice variant, whereby the first splice variant is captured on a first subset of particles, and at most n-1 of the capture extenders are hybridized to the second splice variant, whereby the second splice variant is not captured on the first subset of particles.

0134A related general class of embodiments provides a composition comprising 10 or more subsets of particles, 10 or more subsets of n capture extenders, wherein n is at least two, 10 or more subsets of one or more label extenders, a label probe system comprising a label, and at least a first nucleic acid of interest. A plurality of the particles in each subset are distinguishable from a plurality of the particles in every other subset. (Typically, substantially all of the particles in each subset are distinguishable from substantially all of the particles in every other subset.) The particles in each subset have associated therewith a different capture probe. Each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected subset of the particles. In this class of embodiments, the composition is maintained at a hybridization temperature, which hybridization temperature is greater than a melting temperature T_m of a complex between each individual capture extender and its corresponding capture probe. The first nucleic acid of interest is hybridized to a first subset of n first capture extenders, which first capture extenders are hybridized to a first capture probe.

0135Essentially all of the features noted for the embodiments above apply to these embodiments as well, as relevant; for example, with respect to number of capture extenders per subset, composition of the label probe system, type of label, inclusion of blocking probes, configuration of the capture extenders, capture probes, label extenders, and/or blocking probes, number of nucleic acids of interest and of subsets of particles, capture extenders and label extenders, source of the sample and/or nucleic acids, and/or the like.

0136Another general class of embodiments provides a composition for detecting 10 or more nucleic acids of interest, the composition that includes a solid support comprising 10 or more capture probes, each of which is provided at a selected position on the solid support, 10 or more subsets of n capture extenders, wherein n is at least two, 10 or more subsets of one or more label extenders, and a label probe system comprising a label. Each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected position on the solid support.

0137The composition optionally includes a sample comprising or suspected of comprising at least one of the nucleic acids of interest, e.g., two or more, three or more, etc. nucleic acids. In one class of embodiments, the composition includes at least a first nucleic acid of interest and is maintained at a hybridization temperature. The first nucleic acid of interest is hybridized to a first subset of n first capture extenders, which first capture extenders are hybridized to a first capture probe; the first nucleic acid is thereby associated with a first selected position on the solid support. It will be evident that the composition optionally includes second, third, etc. nucleic acids of interest, which are likewise associated with second, third, etc. selected positions on the solid support through association with second, third, etc. subsets of capture extenders and second, third, etc. capture probes. The hybridization temperature is greater than a melting temperature T_m of a complex between each individual capture extender and its corresponding capture probe. The hybridization temperature is typically about 5°C or more greater than the T_m, e.g., about 7°C or more, about 10°C or more, about 12°C or more, about 15°C or more, about 17°C or more, or even about 20°C or more greater than the T_m.

0138Essentially all of the features noted for the embodiments above apply to these embodiments as well, as relevant; for example, with respect to number of capture extenders per subset, type of solid support, composition of the label probe system, type of label, inclusion of blocking probes, configuration of the capture extenders, capture probes, label extenders, and/or blocking probes, number of nucleic acids of interest and of selected positions on the solid support, capture extenders and label extenders, source of the sample and/or nucleic acids, and/or the like.

KITS

0139A kit for detecting two or more nucleic acids of interest is discussed herein. The kit includes two or more subsets of particles, two or more subsets of n capture extenders, wherein n is at least two, two or more subsets of one or more label extenders, and a label probe system comprising a label, packaged in one or more containers. A plurality of the particles in each subset are distinguishable from a plurality of the particles in every other subset. (Typically, substantially all of the particles in each subset are distinguishable from substantially all of the particles in every other subset.) The particles in each subset have associated therewith a different capture probe. Each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected subset of the particles. When the nucleic acid of interest corresponding to a subset of n capture extenders is hybridized to the subset of n capture extenders, which are hybridized to the corresponding capture probe, the nucleic acid of interest is hybridized to the subset of n capture extenders at a hybridization temperature which is greater than a melting temperature T_m of a complex between each individual capture extender and the capture probe. The kit optionally also includes instructions for using the kit to capture and detect the nucleic acids of interest, one or more buffered solutions (e.g., lysis buffer, diluent, hybridization buffer, and/or wash buffer), standards comprising one or more nucleic acids at known concentration, and/or the like.

0140Essentially all of the features noted for the embodiments above apply to the kits as well, as relevant; for example, with respect to number of capture extenders per subset, composition of the label probe system, type of label, inclusion of blocking probes, configuration of the capture extenders, capture probes, label extenders, and/or blocking probes, number of nucleic acids of interest and of subsets of particles, capture extenders and label extenders, source of the sample and/or nucleic acids, and/or the like.

0141A related kit for detecting two or more nucleic acids of interest is discussed herein. The kit includes a solid support comprising two or more capture probes, each of which is provided at a selected position on the solid support, two or more subsets of n capture extenders, wherein n is at least two, two or more subsets of one or more label extenders, and a label probe system comprising a label, packaged in one or more containers. Each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected position on the solid support.

0142In one class of kits, when a nucleic acid of interest corresponding to a subset of n capture extenders is hybridized to the subset of n capture extenders, which are hybridized to the corresponding capture probe, the nucleic acid of interest is hybridized to the subset of n capture extenders at a hybridization temperature which is greater than a melting temperature T_m of a complex between each individual capture extender and the capture probe. The hybridization temperature is typically about 5°C or more greater than the T_m, e.g., about 7°C or more, about 10°C or more, about 12°C or more, about 15°C or more, about 17°C or more, or even about 20°C or more greater than the T_m.

0143The kit optionally also includes instructions for using the kit to capture and detect the nucleic acids of interest, one or more buffered solutions (e.g., lysis buffer, diluent, hybridization buffer, and/or wash buffer), standards comprising one or more nucleic acids at known concentration, and/or the like.

0144Essentially all of the features noted for the embodiments above apply to these kits as well, as relevant; for example, with respect to number of capture extenders per subset, composition of the label probe system, type of label, inclusion of blocking probes, configuration of the capture extenders, capture probes, label extenders, and/or blocking probes, number of nucleic acids of interest and of selected positions on the solid support, capture extenders and label extenders, source of the sample and/or nucleic acids, and/or the like.

SYSTEMS

0145Discussed herein are systems, e.g., systems used to practice the methods herein and/or comprising the compositions described herein. The system can include, e.g., a fluid and/or microsphere handling element, a fluid and/or microsphere containing element, a laser for exciting a fluorescent label and/or fluorescent microspheres, a detector for detecting light emissions from a chemiluminescent reaction or fluorescent emissions from a fluorescent label and/or fluorescent microspheres, and/or a robotic element that moves other components of the system from place to place as needed (e.g., a multiwell plate handling element). For example, in one class of embodiments, a composition of the invention is contained in a flow cytometer, a Luminex 100^™ or HTS^™ instrument, a microplate reader, a microarray reader, a luminometer, a colorimeter, or like instrument.

0146The system can optionally include a computer. The computer can include appropriate software for receiving user instructions, either in the form of user input into a set of parameter fields, e.g., in a GUI, or in the form of preprogrammed instructions, e.g., preprogrammed for a variety of different specific operations. The software optionally converts these instructions to appropriate language for controlling the operation of components of the system (e.g., for controlling a fluid handling element, robotic element and/or laser). The computer can also receive data from other components of the system, e.g., from a detector, and can interpret the data, provide it to a user in a human readable format, or use that data to initiate further operations, in accordance with any programming by the user.

LABELS

0147A wide variety of labels are well known in the art and can be adapted to the practice of the present invention. For example, luminescent labels and light-scattering labels (e.g., colloidal gold particles) have been described See, e.g., <nplcit id="ncit0025" npl-type="s"><text>Csaki et al. (2002) "Gold nanoparticles as novel label for DNA diagnostics" Expert Rev Mol Diagn 2:187-93</text></nplcit>.

0148As another example, a number of fluorescent labels are well known in the art, including but not limited to, hydrophobic fluorophores (e.g., phycoerythrin, rhodamine, Alexa Fluor 488 and fluorescein), green fluorescent protein (GFP) and variants thereof (e.g., cyan fluorescent protein and yellow fluorescent protein), and quantum dots. See e.g., <nplcit id="ncit0026" npl-type="b"><text>Haughland (2003) Handbook of Fluorescent Probes and Research Products, Ninth Edition or Web Edition, from Molecular Probes, Inc.</text></nplcit>, or <nplcit id="ncit0027" npl-type="b"><text>The Handbook: A Guide to Fluorescent Probes and Labeling Technologies, Tenth Edition or Web Edition (2006) from Invitrog</text></nplcit>en (available on the world wide web at probes.invitrogen.com/handbook) for descriptions of fluorophores emitting at various different wavelengths (including tandem conjugates of fluorophores that can facilitate simultaneous excitation and detection of multiple labeled species). For use of quantum dots as labels for biomolecules, see e.g., <nplcit id="ncit0028" npl-type="s"><text>Dubertret et al. (2002) Science 298:1759</text></nplcit>; <nplcit id="ncit0029" npl-type="s"><text>Nature Biotechnology (2003) 21:41-46</text></nplcit>; and <nplcit id="ncit0030" npl-type="s"><text>Nature Biotechnology (2003) 21:47-51</text></nplcit>.

0149Labels can be introduced to molecules, e.g. polynucleotides, during synthesis or by postsynthetic reactions by techniques established in the art; for example, kits for fluorescently labeling polynucleotides with various fluorophores are available from Molecular Probes, Inc. ((www.) molecularprobes.com), and fluorophore-containing phosphoramidites for use in nucleic acid synthesis are commercially available. Similarly, signals from the labels (e.g., absorption by and/or fluorescent emission from a fluorescent label) can be detected by essentially any method known in the art. For example, multicolor detection, detection of FRET, fluorescence polarization, and the like, are well known in the art.

MICROSPHERES

0150Microspheres are preferred particles in certain embodiments described herein since they are generally stable, are widely available in a range of materials, surface chemistries and uniform sizes, and can be fluorescently dyed. Microspheres can be distinguished from each other by identifying characteristics such as their size (diameter) and/or their fluorescent emission spectra, for example.

0151Luminex Corporation ((www.) luminexcorp.com), for example, offers 100 sets of uniform diameter polystyrene microspheres. The microspheres of each set are internally labeled with a distinct ratio of two fluorophores. A flow cytometer or other suitable instrument can thus be used to classify each individual microsphere according to its predefined fluorescent emission ratio. Fluorescently-coded microsphere sets are also available from a number of other suppliers, including Radix Biosolutions ((www.) radixbiosolutions.com) and Upstate Biotechnology ((www.) upstatebiotech.com). Alternatively, BD Biosciences ((www.) bd.com) and Bangs Laboratories, Inc. ((www.) bangslabs.com) offer microsphere sets distinguishable by a combination of fluorescence and size. As another example, microspheres can be distinguished on the basis of size alone, but fewer sets of such microspheres can be multiplexed in an assay because aggregates of smaller microspheres can be difficult to distinguish from larger microspheres.

0152Microspheres with a variety of surface chemistries are commercially available, from the above suppliers and others (e.g., see additional suppliers listed in <nplcit id="ncit0031" npl-type="s"><text>Kellar and Iannone (2002) "Multiplexed microsphere-based flow cytometric assays" Experimental Hematology 30:1227-1237</text></nplcit> and <nplcit id="ncit0032" npl-type="s"><text>Fitzgerald (2001) "Assays by the score" The Scientist 15[11]:25</text></nplcit>). For example, microspheres with carboxyl, hydrazide or maleimide groups are available and permit covalent coupling of molecules (e.g., polynucleotide capture probes with free amine, carboxyl, aldehyde, sulfhydryl or other reactive groups) to the microspheres. As another example, microspheres with surface avidin or streptavidin are available and can bind biotinylated capture probes; similarly, microspheres coated with biotin are available for binding capture probes conjugated to avidin or streptavidin. In addition, services that couple a capture reagent of the customer's choice to microspheres are commercially available, e.g., from Radix Biosolutions ((www.) radixbiosolutions.com).

0153Protocols for using such commercially available microspheres (e.g., methods of covalently coupling polynucleotides to carboxylated microspheres for use as capture probes, methods of blocking reactive sites on the microsphere surface that are not occupied by the polynucleotides, methods of binding biotinylated polynucleotides to avidin-functionalized microspheres, and the like) are typically supplied with the microspheres and are readily utilized and/or adapted by one of skill. In addition, coupling of reagents to microspheres is well described in the literature. For example, see <nplcit id="ncit0033" npl-type="s"><text>Yang et al. (2001) "BADGE, Beads Array for the Detection of Gene Expression, a high-throughput diagnostic bioassay" Genome Res. 11:1888-98</text></nplcit>; <nplcit id="ncit0034" npl-type="s"><text>Fulton et al. (1997) "Advanced multiplexed analysis with the FlowMetrix™ system" Clinical Chemistry 43:1749-1756</text></nplcit>; <nplcit id="ncit0035" npl-type="s"><text>Jones et al. (2002) "Multiplex assay for detection of strain-specific antibodies against the two variable regions of the G protein of respiratory syncytial virus" 9:633-638</text></nplcit>; <nplcit id="ncit0036" npl-type="s"><text>Camilla et al. (2001) "Flow cytometric microsphere-based immunoassay: Analysis of secreted cytokines in whole-blood samples from asthmatics" Clinical and Diagnostic Laboratory Immunology 8:776-784</text></nplcit>; <nplcit id="ncit0037" npl-type="s"><text>Martins (2002) "Development of internal controls for the Luminex instrument as part of a multiplexed seven-analyte viral respiratory antibody profile" Clinical and Diagnostic Laboratory Immunology 9:41-45</text></nplcit>; <nplcit id="ncit0038" npl-type="s"><text>Kellar and Iannone (2002) "Multiplexed microsphere-based flow cytometric assays" Experimental Hematology 30:1227-1237</text></nplcit>; <nplcit id="ncit0039" npl-type="s"><text>Oliver et al. (1998) "Multiplexed analysis of human cytokines by use of the FlowMetrix system" Clinical Chemistry 44:2057-2060</text></nplcit>; <nplcit id="ncit0040" npl-type="s"><text>Gordon and McDade (1997) "Multiplexed quantification of human IgG, IgA, and IgM with the FlowMetrix™ system" Clinical Chemistry 43:1799-1801</text></nplcit>; <patcit id="pcit0034" dnum="USPN5981180A"><text>USPN 5,981,180</text></patcit> entitled "Multiplexed analysis of clinical specimens apparatus and methods" to Chandler et al. (November 9, 1999); <patcit id="pcit0035" dnum="USPN6449562A"><text>USPN 6,449,562</text></patcit> entitled "Multiplexed analysis of clinical specimens apparatus and methods" to Chandler et al. (September 10, 2002); and references therein.

0154Methods of analyzing microsphere populations (e.g. methods of identifying microsphere subsets by their size and/or fluorescence characteristics, methods of using size to distinguish microsphere aggregates from single uniformly sized microspheres and eliminate aggregates from the analysis, methods of detecting the presence or absence of a fluorescent label on the microsphere subset, and the like) are also well described in the literature. See, e.g., the above references.

0155Suitable instruments, software, and the like for analyzing microsphere populations to distinguish subsets of microspheres and to detect the presence or absence of a label (e.g., a fluorescently labeled label probe) on each subset are commercially available. For example, flow cytometers are widely available, e.g., from Becton-Dickinson ((www.) bd.com) and Beckman Coulter ((www.) beckman.com). Luminex 100^™ and Luminex HTS^™ systems (which use microfluidics to align the microspheres and two lasers to excite the microspheres and the label) are available from Luminex Corporation ((www.) luminexcorp.com); the similar Bio-Plex^™ Protein Array System is available from Bio-Rad Laboratories, Inc. ((www.) bio-rad.com). A confocal microplate reader suitable for microsphere analysis, the FMAT^™ System 8100, is available from Applied Biosystems ((www.) appliedbiosystems.com).

0156As another example of particles that can be adapted for use in the present invention, sets of microbeads that include optical barcodes are available from CyVera Corporation ((www.) cyvera.com). The optical barcodes are holographically inscribed digital codes that diffract a laser beam incident on the particles, producing an optical signature unique for each set of microbeads.

MOLECULAR BIOLOGICAL TECHNIQUES

0157In practicing the present invention, many conventional techniques in molecular biology, microbiology, and recombinant DNA technology are optionally used. These techniques are well known and are explained in, for example, <nplcit id="ncit0041" npl-type="b"><text>Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA</text></nplcit>; <nplcit id="ncit0042" npl-type="b"><text>Sambrook et al., Molecular Cloning - A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2000</text></nplcit> and <nplcit id="ncit0043" npl-type="b"><text>Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley &amp; Sons, Inc., (supplemented through 2005</text></nplcit>). Other useful references, e.g. for cell isolation and culture (e.g., for subsequent nucleic acid or protein isolation) include <nplcit id="ncit0044" npl-type="b"><text>Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York</text></nplcit> and the references cited therein; <nplcit id="ncit0045" npl-type="b"><text>Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &amp; Sons, Inc. New York, NY</text></nplcit>; <nplcit id="ncit0046" npl-type="b"><text>Gamborg and Phillips (Eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York</text></nplcit>) and <nplcit id="ncit0047" npl-type="b"><text>Atlas and Parks (Eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL</text></nplcit>.

Making Polynucleotides

0158Methods of making nucleic acids (e.g., by in vitro amplification, purification from cells, or chemical synthesis), methods for manipulating nucleic acids (e.g., by restriction enzyme digestion, ligation, etc.) and various vectors, cell lines and the like useful in manipulating and making nucleic acids are described in the above references. In addition, methods of making branched polynucleotides (e.g., amplification multimers) are described in USPN 5,635,352, USPN 5,124,246, USPN 5,710,264, and USPN 5,849,481, as well as in other references mentioned above.

0159In addition, essentially any polynucleotide (including, e.g., labeled or biotinylated polynucleotides) can be custom or standard ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company ((www.) mcrc.com), The Great American Gene Company ((www.) genco.com), ExpressGen Inc. ((www.) expressgen.com), Qiagen (oligos.qiagen.com) and many others.

0160A label, biotin, or other moiety can optionally be introduced to a polynucleotide, either during or after synthesis. For example, a biotin phosphoramidite can be incorporated during chemical synthesis of a polynucleotide. Alternatively, any nucleic acid can be biotinylated using techniques known in the art; suitable reagents are commercially available, e.g., from Pierce Biotechnology ((www.) piercenet.com). Similarly, any nucleic acid can be fluorescently labeled, for example, by using commercially available kits such as those from Molecular Probes, Inc. ((www.) molecularprobes.com) or Pierce Biotechnology ((www.) piercenet.com) or by incorporating a fluorescently labeled phosphoramidite during chemical synthesis of a polynucleotide.

ARRAYS

0161In an array of capture probes on a solid support (e.g., a membrane, a glass or plastic slide, a silicon or quartz chip, a plate, or other spatially addressable solid support), each capture probe is typically bound (e.g., electrostatically or covalently bound, directly or via a linker) to the support at a unique selected location. Methods of making, using, and analyzing such arrays (e.g., microarrays) are well known in the art. See, e.g., <nplcit id="ncit0048" npl-type="b"><text>Baldi et al. (2002) DNA Microarrays and Gene Expression: From Experiments to Data Analysis and Modeling, Cambridge University Press</text></nplcit>; <nplcit id="ncit0049" npl-type="s"><text>Beaucage (2001) "Strategies in the preparation of DNA oligonucleotide arrays for diagnostic applications" Curr Med Chem 8:1213-1244</text></nplcit>; <nplcit id="ncit0050" npl-type="b"><text>Schena, ed. (2000) Microarray Biochip Technology, pp.19-38, Eaton Publishing; technical note "Agilent SurePrint Technology: Content centered microarray design enabling speed and flexibility" available on the web at chem.agilent.com/temp/rad01539/00039489.pdf</text></nplcit>; and references therein. Arrays of pre-synthesized polynucleotides can be formed (e.g., printed), for example, using commercially available instruments such as a GMS 417 Arrayer (Affymetrix, Santa Clara, CA). Alternatively, the polynucleotides can be synthesized at the selected positions on the solid support; see, e.g., USPN 6,852,490 and USPN 6,306,643, each to Gentanlen and Chee entitled "Methods of using an array of pooled probes in genetic analysis."

0162Suitable solid supports are commercially readily available. For example, a variety of membranes (e.g., nylon, PVDF, and nitrocellulose membranes) are commercially available, e.g., from Sigma-Aldrich, Inc. ((www.) sigmaaldrich.com). As another example, surface-modified and pre-coated slides with a variety of surface chemistries are commercially available, e.g., from TeleChem International ((www.) arrayit.com), Corning, Inc. (Corning, NY), or Greiner Bio-One, Inc. ((www.) greinerbiooneinc.com). For example, silanated and silyated slides with free amino and aldehyde groups, respectively, are available and permit covalent coupling of molecules (e.g., polynucleotides with free aldehyde, amine, or other reactive groups) to the slides. As another example, slides with surface streptavidin are available and can bind biotinylated capture probes. In addition, services that produce arrays of polynucleotides of the customer's choice are commercially available, e.g., from TeleChem International ((www.) arrayit.com) and Agilent Technologies (Palo Alto, CA).

0163Suitable instruments, software, and the like for analyzing arrays to distinguish selected positions on the solid support and to detect the presence or absence of a label (e.g., a fluorescently labeled label probe) at each position are commercially available. For example, microarray readers are available, e.g., from Agilent Technologies (Palo Alto, CA), Affymetrix (Santa Clara, CA), and Zeptosens (Switzerland).

EXAMPLES

0164It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art. Accordingly, the following examples are offered to illustrate, but not to limit, the claimed invention.

<u>EXAMPLE 1: A MULTIPLEX BRANCHED DNA ASSAY FOR PARALLEL QUANTITATIVE GENE EXPRESSION PROFILING</u>

0165The following sets forth a series of experiments that demonstrate detection of nucleic acids using a multiplex bDNA assay, in which the nucleic acids are captured to distinct subsets of microspheres through cooperative hybridization. This example describes a new method to quantitatively measure the expression of multiple mRNAs directly from crude cell lysates and tissue homogenates without the need for RNA purification or target amplification. A distinguishing feature of this multiplex bDNA assay is the use of cooperative hybridization (a strong, stable hybridization interaction formed by the collective force of multiple weak, unstable hybridization interactions) in the assay design, which ensures an exceptionally high degree of assay specificity. The experiments demonstrate that the assay is capable of detecting a single transcript of mRNA per cell from as few as 25,000 cells and with intra-plate and inter-plate coefficients of variance (CV) of less than 10% and 15%, respectively. To demonstrate the utility of this technology, several 10-plex (ten-gene) panels were developed to quantify the expression of genes involved in the proinflammatory and apoptosis stimulation pathways. The data from the multiplex bDNA assay was compared with that of single-plex bDNA assays with a high degree of correlation. The multiplex bDNA assay thus provides a powerful means to quantify the gene expression profile of a defined set of target genes in large sample populations.

0166As described above, the branched DNA (bDNA) assay provides a useful approach for the quantification of mRNA transcripts. A significant departure from target amplification methods, the bDNA assay can directly measure mRNA from crude cell lysates and tissue homogenates by amplifying the reporter signal, and thus avoiding the errors inherent in the extraction and amplification of target sequences. Owing to its robustness and precision, the bDNA assay has been used in clinical diagnostic applications such as viral load determination, and in drug discovery research studying drug metabolism, structure-activity relationships, high throughput screens, and more recently, siRNA knockdown analysis (<nplcit id="ncit0051" npl-type="s"><text>Urdea et al. (1991) "Branched DNA amplification multimers for the sensitive, direct detection of human hepatitis virus" Nuc Acids Symp Ser 24:197-200</text></nplcit>, <nplcit id="ncit0052" npl-type="s"><text>Gleaves et al. (2002) "Multicenter evaluation of the Bayer VERSANT HIV-1 RNA 3.0 assay: analytical and clinical performance" J Clin Virol. 25:205-16</text></nplcit>, <nplcit id="ncit0053" npl-type="s"><text>Hartley and Klaassen (2000) "Detection of chemical-induced differential expression of rat hepatic cytochrome P450 mRNA transcripts using branched DNA signal amplification technology" Drug Metab Dispos. 28:608-16</text></nplcit>, <nplcit id="ncit0054" npl-type="s"><text>Bramlett et al. (2000) "Correlation of farnesoid X receptor coactivator recruitment and cholesterol 7alpha-hydroxylase gene repression by bile acids" Mol Genet Metab. 71:609-15</text></nplcit>, and <nplcit id="ncit0055" npl-type="s"><text>Warrior et al. (2000) "Application of QuantiGene nucleic acid quantification technology for high throughput screening" J Biomol Screen. 5:343-52</text></nplcit>, and <nplcit id="ncit0056" npl-type="s"><text>Soutschek et al. (2004) "Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs" Nature 432:173-8</text></nplcit>).

0167The Luminex bead-based array system has been used in a wide range of multiplex applications throughout the drug-discovery and diagnostics fields, as well as basic research (<nplcit id="ncit0057" npl-type="s"><text>Iannone (2000) "Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry" Cytometry 39:131-40</text></nplcit>, <nplcit id="ncit0058" npl-type="s"><text>Yang et al. (2001) "BADGE, Beads Array for the Detection of Gene Expression, a high-throughput diagnostic bioassay" Genome Res. 11:1888-98</text></nplcit>, and <nplcit id="ncit0059" npl-type="s"><text>Fulton et al. (1997) "Advanced multiplexed analysis with the FlowMetrix system" Clin Chem. 43:1749-56</text></nplcit>). For example, it has been a widely adopted platform for quantitative multiplex protein expression analysis. At the core are 100 fluorescent-encoded microsphere beads that can be coupled with a capture reagent specific to a particular bioassay, allowing for the detection of up to 100 unique analytes within a single sample.

0168A multiplex bDNA assay has been developed that combines the advantages of the conventional single-plex bDNA assay with, e.g., the multiplex capability of the Luminex platform. The fluorescent beads are coupled with a set of oligonucleotide capture probes, and cooperative hybridization is exploited in the assay design for exceptionally high assay specificity. The new mRNA quantification method measures the expression levels of multiple mRNA transcripts quantitatively from purified RNA and crude cell lysates with high accuracy and reproducibility. The simplicity and data quality of the assay makes it an ideal tool for high throughput parallel quantitative gene expression analysis.

Results

Overview of the multiplex bDNA assay

0169In the multiplex bDNA assay, fluorescent-encoded microsphere beads (Luminex Corporation) were used for the capture of specific mRNA species (<figref idref="f0007"><b>Figure 5</b></figref><b>Panels A-C</b>). The ability to quantify multiple mRNA transcripts lies in the design of a probe set for each mRNA transcript of interest. A probe set contains three types of synthetic oligonucleotide probes called capture extenders (CEs, <b>501-503</b>), label extenders (LEs, <b>506-508</b>), and blockers (BPs, <b>510</b>) that hybridize and span a contiguous region of the target mRNA (<b>511-513</b>, provided for example in a cell lysate or tissue homogenate). The intended target is captured by multiple CEs, which hybridize specifically to their complementary capture probes (CPs, <b>521-523</b>) bound to the fluorescent-encoded beads (<b>526-528</b>) and allow the target mRNA to be captured to its corresponding beads. Signal amplification occurs when the LE tails are hybridized with a branched DNA molecule (bDNA amplifier, <b>530),</b> which can then be hybridized with biotinylated label probes <b>(531)</b> followed by streptavidin-conjugated phycoerythrin (SAPE, <b>532).</b> Bead fluorescent color codes and SAPE reporter signals from all beads in the final hybridization mixture are determined using a Luminex flow cytometer (or similar instrument), which maps each bead to a specific mRNA assay and provides a fluorescence measurement of SAPE reporter associated with that bead. The SAPE signals are proportional to the number of mRNA transcripts captured to the beads.

0170The assay includes three major steps: (1) Capture of the specific mRNA transcripts to their corresponding beads through CE-CP interaction during an overnight hybridization at 53°C <b>(</b><figref idref="f0007"><b>Figure 5</b></figref><b>Panel A);</b> (2) Hybridization with the bDNA amplification molecule and biotinylated label probe, respectively, for an hour at 46°C <b>(</b><figref idref="f0007"><b>Figure 5</b></figref><b>Panel B);</b> and (3) Hybridization with streptavidin-conjugated R-phycoerythrin (SAPE) at room temperature for 30 minutes <b>(</b><figref idref="f0007"><b>Figure 5</b></figref><b>Panel C).</b> The Luminex beads are then analyzed with a Luminex 100^™ system. The level of SAPE fluorescence measured from each bead is proportional to the amount of mRNA transcript captured by the bead.

Cooperative hybridization

0171When the melting temperature (T_m) of an oligonucleotide duplex (e.g., a CP-CE hybridization interaction) is below the actual hybridization temperature, dissociation to its single-stranded form is favored under the assay condition. Such single hybridization does not result in stable capture of the target mRNA under this condition. However, when multiple weak hybridization interactions exist, the collective force of these weak hybridization allows the stable capture of the target mRNA. <figref idref="f0008"><b>Figure 6</b></figref><b>Panels A</b> and <b>B</b> depict simple and cooperative hybridization, respectively. Simple hybridization between a CP and CE under the standard bDNA assay conditions is illustrated by the results shown in <figref idref="f0008"><b>Figure 6</b></figref><b>Panel C.</b> CP14, CP15, and CP16 represent capture probes where the complementary sequence between CP and CE are 14, 15, or 16 bases in length, respectively. CP-N represents a nonspecific capture probe that does not hybridize with the CE sequence. The CE-LE represents a probe that can bind to the CP at one end and the bDNA amplification molecule at the other end. The CE represents a probe that binds to CP only but not the bDNA amplification molecule. Cooperative hybridization between CP and CE under the standard bDNA assay conditions is illustrated by the results shown in <figref idref="f0008"><b>Figure 6</b></figref><b>Panel D.</b> A standard single-plex bDNA assay on IL-6 IVT RNA dilution series was performed in assay plate wells where CP14, CP15, or CP16 was attached to the surface, respectively. RLU stands for the Relative Luminescent Unit and the error bars represent 1 s.d.

0172A strong, stable hybridization interaction can be produced through the joint force of multiple weak, unstable hybridization interactions. The weak, unstable hybridization interaction occurs when the melting temperature (T_m) of the hybridizing nucleic acids is below the assay temperature. This concept is termed cooperative hybridization. Cooperative hybridization can be applied in bDNA assays to allow highly specific capture of target mRNA. When simple hybridization (<figref idref="f0008"><b>Figure 6</b></figref><b>Panel A</b>) between the CE and the CP occurs at a temperature above their melting temperature, the target mRNA cannot be stably captured to the solid surface. However, when multiple of those weak CE-CP hybridizations occur at the same time (<figref idref="f0008"><b>Figure 6</b></figref><b>Panel B</b>), the target mRNA can be stably captured to the solid surface. To demonstrate cooperative hybridization, three capture probes varying in length from 14mer to 16mer were designed and used in a plate-based, single-plex bDNA assay. The 14 to 16 base capture probes were chosen because the melting temperature (T_m) is below the hybridization temperature of the bDNA assay, which is at 53°C. The simple hybridization between the CP and the CE was evaluated through an oligonucleotide (CE-LE) that contains a CE tail sequence complementary to the capture probe and an LE tail sequence complementary to a sequence in the bDNA amplifier molecule. A strong assay signal is obtained when the capture probe is 16 bases in length, a 100-fold weaker hybridization interaction is evident when the capture probe is 15 bases in length, and minimal hybridization interaction occurs when the capture probe is 14 bases in length (<figref idref="f0008"><b>Figure 6</b></figref><b>Panel C</b>). When the three capture probes were used respectively in a bDNA assay where multiple CEs were present, overlapping assay signal was obtained regardless of capture probe length (<figref idref="f0008"><b>Figure 6</b></figref><b>Panel D</b>). This suggests that cooperative hybridization plays a prominent role in the bDNA assay, especially when the capture probe is 14 bases long.

0173Using the cooperative hybridization concept, a set of capture probes that work together in a single hybridization reaction with minimal nonspecific cross-hybridization was designed. The capture probes were designed to comprise unique 15mer DNA sequences and a common linker that allows them to be chemically cross-linked onto the surface of the different fluorescent-encoded bead populations. Each bead population was examined for specific hybridization to its complementary biotinylated oligonucleotide as well as for its non-specific hybridization to the other biotinylated oligonucleotides. The assay signals of all possible non-specific hybridizations were less than 0.1 % of those observed for the perfectly matched pair, indicating a very high degree of hybridization specificity. Thus the set of capture probes was shown to be highly specific and can be used in the multiplex bDNA assay for the capture of target mRNA.

Performance evaluation of multiplex bDNA assay

0174To demonstrate the performance of the multiplex bDNA assay, several 10-plex panels were developed (three of which are described in <b>Table 1</b>) and evaluated for sensitivity, linear dynamic range, precision and specificity using in vitro transcribed (IVT) RNA transcripts as reference standards. The performance evaluation data for the cytokine panel (<figref idref="f0009 f0010"><b>Figure 7</b></figref> and <b>Table 2</b>) is below. Probe sets for the cytokine panel are listed in <b>Table 3.</b>

0175To determine the cross-reactivity between target mRNAs, 40 amol of each IVT RNA transcript was individually hybridized into the bead array followed by measurement of signals for the intended target as well as for other genes. Cross-reactivity is expressed as the percentage of the average signal for other genes over the signal for the intended target. When 40 amol of individual mRNA transcripts (represented by gene name on the x-axis of <figref idref="f0009 f0010"><b>Figure 7</b></figref><b>Panel A</b>) was added into the fluorescent-encoded bead array, only the corresponding bead (represented by number on the y-axis) gives a strong fluorescent signal (z-axis). Net MFI stands for background subtracted median fluorescent intensity from 100 counted beads. The cross-reactivity between target genes in the panel was less than 0.1%, suggesting minimal cross hybridization between the genes in the panel (<figref idref="f0009 f0010"><b>Figure 7</b></figref><b>Panel A and Table 2</b>).

0176The assay specificity was evaluated by measuring the signal of <i>B. subtilis</i> dapB IVT RNA, which has little homology with human RNA, and human IL10 IVT RNA, which has undetectable expression in human U937 cells, as model genes. The IVT RNA transcripts of dapB and IL10 were serially diluted four-fold from 160 to 0.04 amol and mixed with 0.2 µg of total RNA extracted from human U937 cells. A multiplex bDNA assay was performed and the assay signals for dapB (<figref idref="f0009 f0010"><b>Figure 7</b></figref><b>Panel B</b>) and IL10 (<figref idref="f0009 f0010"><b>Figure 7</b></figref><b>Panel C</b>) were compared in the presence (squares) or absence (diamonds) of the U937 total RNA background. Overlapping signals were observed through the entire dilution series for both dapB and IL10, suggesting that minimal nonspecific hybridization occurred. Importantly, the addition of 0.2 µg of total RNA did not increase the assay background, further demonstrating the assay specificity. Tests that determine spike recovery of a target from a complex mixture are a typically recommended analytical procedure to assess assay accuracy. The assay is considered reliable if the signal difference between pure and spiked-in analyte is within ±20%. In this case, no change in assay signal was observed in the presence of U937 total RNA, indicating that nonspecific RNA does not seem to interfere with the hybridization of the target RNA to the capture beads. As a result, IVT RNA standard curves can be used to quantify the absolute number of copies of RNA transcripts in a sample.

0177To determine the assay sensitivity and linear dynamic range, ten target IVT RNA transcripts were equally mixed and serially diluted four-fold to generate standard curves with target RNA levels ranging from 2.4x 10⁴ to 9.6x 16⁷ transcripts (0.04 to 160 amol). The signal responses for all the cytokine genes were linear across the target concentration range examined with coefficients of correlation (R²) averaged 0.99, indicating that the linear dynamic range of the assay spans more than 3 logs (<figref idref="f0009 f0010"><b>Figure 7</b></figref><b>Panel D</b>). The sensitivity of the assay was evaluated by determining the limit of detection (LOD) for each target RNA. LOD, defined as the target concentration at which the signal is three standard deviations above the background, is 0.06 amol for VEGF and 0.04 amol for the remaining cytokines (<b>Table 2</b>).

0178Assay precision between different wells (intra-plate) and between assays performed on different days (inter-plate) was assessed by calculating coefficients of variation (%CV) for each gene expression measurement from 0.04 -160 amol IVT across multiple samples. Precision value for each gene was measured across multiple wells within a single plate (n = 4) and across multiple plates (N = 3). Intra-plate CV averaged about 8%, ranging from 5 to 14%, and inter-plate CV averaged about 14%, ranging from 7% to 22% (<b>Table 2</b>). Average intra-plate CV of less than 10% has been routinely obtained for multiplex bDNA assays with triplicate samples. It should be noted that the CVs were highly comparable from high to low concentrations of the target IVT RNA tested, suggesting that accurate quantification can be achieved in a target concentration-independent fashion.

0179<tables id="tabl0001" num="0001"><table frame="all"><title><b>Table 1.</b> Target names and reference sequence accession numbers for the cytokine and apoptosis 10-plex panels.</title><tgroup cols="7"><colspec colnum="1" colname="col1" colwidth="23mm" /><colspec colnum="2" colname="col2" colwidth="24mm" /><colspec colnum="3" colname="col3" colwidth="24mm" /><colspec colnum="4" colname="col4" colwidth="25mm" /><colspec colnum="5" colname="col5" colwidth="24mm" /><colspec colnum="6" colname="col6" colwidth="24mm" /><colspec colnum="7" colname="col7" colwidth="24mm" /><thead><row><entry align="center" valign="top" /><entry namest="col2" nameend="col3" align="center" valign="top">Cytokine Panel</entry><entry namest="col4" nameend="col5" align="center" valign="top">Apoptosis Panel 1</entry><entry namest="col6" nameend="col7" align="center" valign="top">Apoptosis Panel 2</entry></row><row valign="middle"><entry align="center">Bead Number</entry><entry align="center">Target Symbol</entry><entry align="center">Accession Number</entry><entry align="center">Target Symbol</entry><entry align="center">Accession Number</entry><entry align="center">Target Symbol</entry><entry align="center">Accession. Number</entry></row></thead><tbody><row><entry align="center">22</entry><entry align="center">IL2</entry><entry align="center">NM_000586</entry><entry align="center">RELB</entry><entry align="center">NM_006509</entry><entry align="center">BAK1</entry><entry align="center">NM_001188</entry></row><row><entry align="center">29</entry><entry align="center">TNF</entry><entry align="center">NM_000594</entry><entry align="center">TNF</entry><entry align="center">NM_000594</entry><entry align="center">TNFSF6</entry><entry align="center">NM_000639</entry></row><row><entry align="center">19</entry><entry align="center">VEGF</entry><entry align="center">NM_003376</entry><entry align="center">NFKB2</entry><entry align="center">NM_002502</entry><entry align="center">IL6R</entry><entry align="center">NM_000565</entry></row><row><entry align="center">28</entry><entry align="center">IL10</entry><entry align="center">NM_000572</entry><entry align="center">TNFAIP3</entry><entry align="center">NM_006290</entry><entry align="center">TNFRSF6</entry><entry align="center">NM_000043</entry></row><row><entry align="center">25</entry><entry align="center">IL6</entry><entry align="center">NM_000600</entry><entry align="center">IL6</entry><entry align="center">NM_000600</entry><entry align="center">PTK2B</entry><entry align="center">NM_004103</entry></row><row><entry align="center">33</entry><entry align="center">IL1B</entry><entry align="center">NM_000576</entry><entry align="center">CDKN1A</entry><entry align="center">NM_000389</entry><entry align="center">BAD</entry><entry align="center">NM_004322</entry></row><row><entry align="center">20</entry><entry align="center">IFNG</entry><entry align="center">NM_000619</entry><entry align="center">NFKB1</entry><entry align="center">NM_003998</entry><entry align="center">BCL2</entry><entry align="center">NM_000633</entry></row><row><entry align="center">18</entry><entry align="center">IL8</entry><entry align="center">NM_000584</entry><entry align="center">RELA</entry><entry align="center">NM_021975</entry><entry align="center">BCL2L1</entry><entry align="center">NM_138578</entry></row><row><entry align="center">27</entry><entry align="center">CSF2</entry><entry align="center">NM_000758</entry><entry align="center">NFKBIA</entry><entry align="center">NM_020529</entry><entry align="center">CFLAR</entry><entry align="center">NM_003879</entry></row><row><entry align="center">26</entry><entry align="center">GAPD</entry><entry align="center">NM_002046</entry><entry align="center">GAPD</entry><entry align="center">NM_002046</entry><entry align="center">ACTB</entry><entry align="center">NM_001101</entry></row><row><entry align="center">34</entry><entry align="center">dapB</entry><entry align="center">L38424</entry><entry align="center">N/A</entry><entry align="center">N/A</entry><entry align="center">N/A</entry><entry align="center">N/A</entry></row></tbody></tgroup></table></tables>

0180<tables id="tabl0002" num="0002"><table frame="all"><title><b>Table 2.</b> Detection sensitivity, cross-reactivity, and assay background of the cytokine 10-plex panel.</title><tgroup cols="12"><colspec colnum="1" colname="col1" colwidth="20mm" /><colspec colnum="2" colname="col2" colwidth="14mm" /><colspec colnum="3" colname="col3" colwidth="14mm" /><colspec colnum="4" colname="col4" colwidth="13mm" /><colspec colnum="5" colname="col5" colwidth="13mm" /><colspec colnum="6" colname="col6" colwidth="13mm" /><colspec colnum="7" colname="col7" colwidth="13mm" /><colspec colnum="8" colname="col8" colwidth="13mm" /><colspec colnum="9" colname="col9" colwidth="13mm" /><colspec colnum="10" colname="col10" colwidth="13mm" /><colspec colnum="11" colname="col11" colwidth="14mm" /><colspec colnum="12" colname="col12" colwidth="15mm" /><thead><row><entry align="center" valign="top">Gene</entry><entry align="center" valign="top">CSF2</entry><entry align="center" valign="top">GAPD</entry><entry align="center" valign="top">IFNG</entry><entry align="center" valign="top">IL10</entry><entry align="center" valign="top">IL1B</entry><entry align="center" valign="top">IL2</entry><entry align="center" valign="top">IL6</entry><entry align="center" valign="top">IL8</entry><entry align="center" valign="top">TNF</entry><entry align="center" valign="top">VEG F</entry><entry align="center" valign="top">dapB</entry></row></thead><tbody><row><entry align="center">LOD (amol)</entry><entry align="center" valign="middle">0.04</entry><entry align="center" valign="middle">0.04</entry><entry align="center" valign="middle">0.04</entry><entry align="center" valign="middle">0.04</entry><entry align="center" valign="middle">0.04</entry><entry align="center" valign="middle">0.04</entry><entry align="center" valign="middle">0.04</entry><entry align="center" valign="middle">0.04</entry><entry align="center" valign="middle">0.04</entry><entry align="center" valign="middle">0.06</entry><entry align="center" valign="middle">0.04</entry></row><row><entry align="center">% Cross-reactivity*</entry><entry align="center" valign="middle">0.004 %</entry><entry align="center" valign="middle">0.003 %</entry><entry align="center" valign="middle">0.006 %</entry><entry align="center" valign="middle">0.007 %</entry><entry align="center" valign="middle">0.004 %</entry><entry align="center" valign="middle">0.006 %</entry><entry align="center" valign="middle">0.018 %</entry><entry align="center" valign="middle">0.100 %</entry><entry align="center" valign="middle">0.002 %</entry><entry align="center" valign="middle">0.139 %</entry><entry align="center" valign="middle">0.018 %</entry></row><row><entry align="center">Average backgroun d(MFI)</entry><entry align="center" valign="middle">2.4</entry><entry align="center" valign="middle">2.3</entry><entry align="center" valign="middle">1.9</entry><entry align="center" valign="middle">3.6</entry><entry align="center" valign="middle">3.7</entry><entry align="center" valign="middle">1.9</entry><entry align="center" valign="middle">2.0</entry><entry align="center" valign="middle">1.1</entry><entry align="center" valign="middle">3.0</entry><entry align="center" valign="middle">15.9</entry><entry align="center" valign="middle">5.5</entry></row><row><entry align="center" valign="middle">% Intra-plate CV</entry><entry align="center" valign="middle">9%</entry><entry align="center" valign="middle">8%</entry><entry align="center" valign="middle">8%</entry><entry align="center" valign="middle">7%</entry><entry align="center" valign="middle">7%</entry><entry align="center" valign="middle">5%</entry><entry align="center" valign="middle">8%</entry><entry align="center" valign="middle">14%</entry><entry align="center" valign="middle">9%</entry><entry align="center" valign="middle">7%</entry><entry align="center" valign="middle">5%</entry></row><row><entry align="center" valign="middle">% Inter-plate CV</entry><entry align="center" valign="middle">21%</entry><entry align="center" valign="middle">12%</entry><entry align="center" valign="middle">11%</entry><entry align="center" valign="middle">10%</entry><entry align="center" valign="middle">22%</entry><entry align="center" valign="middle">7%</entry><entry align="center" valign="middle">13%</entry><entry align="center" valign="middle">10%</entry><entry align="center" valign="middle">18%</entry><entry align="center" valign="middle">20%</entry><entry align="center" valign="middle">7%</entry></row></tbody></tgroup><tgroup cols="12" rowsep="0"><colspec colnum="1" colname="col1" colwidth="20mm" /><colspec colnum="2" colname="col2" colwidth="14mm" /><colspec colnum="3" colname="col3" colwidth="14mm" /><colspec colnum="4" colname="col4" colwidth="13mm" /><colspec colnum="5" colname="col5" colwidth="13mm" /><colspec colnum="6" colname="col6" colwidth="13mm" /><colspec colnum="7" colname="col7" colwidth="13mm" /><colspec colnum="8" colname="col8" colwidth="13mm" /><colspec colnum="9" colname="col9" colwidth="13mm" /><colspec colnum="10" colname="col10" colwidth="13mm" /><colspec colnum="11" colname="col11" colwidth="14mm" /><colspec colnum="12" colname="col12" colwidth="15mm" /><tbody><row><entry namest="col1" nameend="col12" align="justify">*Cross-reactivity is the percentage of signal generated by hybridization of mRNA to nontarget beads, in relation to the specific hybridization to its target bead (100%)</entry></row></tbody></tgroup></table></tables>

0181<tables id="tabl0003" num="0003"><table frame="all"><title><b>Table 3.</b> Probe sets (CPs, CEs, LEs, and BPs) for the cytokine panel.</title><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="14mm" /><colspec colnum="2" colname="col2" colwidth="10mm" /><colspec colnum="3" colname="col3" colwidth="127mm" /><colspec colnum="4" colname="col4" colwidth="15mm" /><thead><row><entry valign="top">Gene</entry><entry valign="top" /><entry valign="top" /><entry valign="top">SEQ ID NO</entry></row></thead><tbody><row><entry>CSF2</entry><entry>BP</entry><entry>GGGCTGGGCGAGCGG</entry><entry>5</entry></row><row><entry>CSF2</entry><entry>BP</entry><entry>CCAGGGCTGCGTGCTG</entry><entry>6</entry></row><row><entry>CSF2</entry><entry>BP</entry><entry>AGACGCCGGGCCTCC</entry><entry>7</entry></row><row><entry>CSF2</entry><entry>BP</entry><entry>CCGCAGGCCCTGCTTG</entry><entry>8</entry></row><row><entry>CSF2</entry><entry>BP</entry><entry>TCATGGTCAAGGGGCCCT</entry><entry>9</entry></row><row><entry>CSF2</entry><entry>BP</entry><entry>GGGGTTGGAGGGCAGTGC</entry><entry>10</entry></row><row><entry>CSF2</entry><entry>BP</entry><entry>CAGCAGTCAAAGGGGATGACA</entry><entry>11</entry></row><row><entry>CSF2</entry><entry>LE</entry><entry>caggccacagtgcccaagTTTTTaggcataggacccgtgtct</entry><entry>12</entry></row><row><entry>CSF2</entry><entry>LE</entry><entry>tggatggcattcacatgctcTTTTTaggcataggacccgtgtct</entry><entry>13</entry></row><row><entry>CSF2</entry><entry>LE</entry><entry>gcagtgtctctactcaggttcaggTTTTTaggcataggacccgtgtct</entry><entry>14</entry></row><row><entry>CSF2</entry><entry>LE</entry><entry>ttctactgtttcattcatctcagcaTTTTTaggcataggacccgtgtct</entry><entry>15</entry></row><row><entry>CSF2</entry><entry>LE</entry><entry>ggaggtcaaacatttctgagatgacTTTTTaggcataggacccgtgtct</entry><entry>16</entry></row><row><entry>CSF2</entry><entry>LE</entry><entry>tgtaggcaggtcggctcctTTTTTaggcataggacccgtgtct</entry><entry>17</entry></row><row><entry>CSF2</entry><entry>LE</entry><entry>gggttgcacaggaagtttccTTTTTaggcataggacccgtgtct</entry><entry>18</entry></row><row><entry>CSF2</entry><entry>LE</entry><entry>tttgaaactttcaaaggtgataatctTTTTTaggcataggacccgtgtct</entry><entry>19</entry></row><row><entry>CSF2</entry><entry>LE</entry><entry>agcagaaagtccttcaggttctcTTTTTaggcataggacccgtgtct</entry><entry>20</entry></row><row><entry>CSF2</entry><entry>LE</entry><entry>ctcactcctggactggctccTTTTTaggcataggacccgtgtct</entry><entry>21</entry></row><row><entry>CSF2</entry><entry>CE</entry><entry>agcagcaggctctgcagcTTTTTttgtgcagtgttata</entry><entry>22</entry></row><row><entry>CSF2</entry><entry>CE</entry><entry>gcgggtgcagagatgctgTTTTTttgtgcagtgttata</entry><entry>23</entry></row><row><entry>CSF2</entry><entry>CE</entry><entry>tacagctccaggcgggtcTTTTTttgtgcagtgttata</entry><entry>24</entry></row><row><entry>CSF2</entry><entry>CE</entry><entry>tgagcttggtgaggctgccTTTTTttgtgcagtgttata</entry><entry>25</entry></row><row><entry>CSF2</entry><entry>CE</entry><entry>tgcttgtagtggctggccaTTTTTttgtgcagtgttata</entry><entry>26</entry></row><row><entry>CSF2</entry><entry>CP</entry><entry>TTTTTTTTTATAACACTGCACAA</entry><entry>27</entry></row><row><entry>GAPD</entry><entry>BP</entry><entry>TCAGCGCCAGCATCGC</entry><entry>28</entry></row><row><entry>GAPD</entry><entry>BP</entry><entry>GAGGGGGCAGAGATGATGAC</entry><entry>29</entry></row><row><entry>GAPD</entry><entry>BP</entry><entry>AACATGGGGGCATCAGCA</entry><entry>30</entry></row><row><entry>GAPD</entry><entry>BP</entry><entry>CATGGTTCACACCCATGACG</entry><entry>31</entry></row><row><entry>GAPD</entry><entry>BP</entry><entry>CGGAGGGGCCATCCAC</entry><entry>32</entry></row><row><entry>GAPD</entry><entry>BP</entry><entry>TGGAGAGCCCCGCGG</entry><entry>33</entry></row><row><entry>GAPD</entry><entry>BP</entry><entry>gcaggaggcattgctgatga</entry><entry>34</entry></row><row><entry>GAPD</entry><entry>LE</entry><entry>gatgggatttccattgatgacaTTTTTaggcataggacccgtgtct</entry><entry>35</entry></row><row><entry>GAPD</entry><entry>LE</entry><entry>cccacttgattttggagggaTTTTTaggcataggacccgtgtct</entry><entry>36</entry></row><row><entry>GAPD</entry><entry>LE</entry><entry>ccagtggactccacgacgtacTTTTTaggcataggacccgtgtct</entry><entry>37</entry></row><row><entry>GAPD</entry><entry>LE</entry><entry>ttctccatggtggtgaagacgTTTTTaggcataggacccgtgtct</entry><entry>38</entry></row><row><entry>GAPD</entry><entry>LE</entry><entry>tcttgaggctgttgtcatacttctTTTTTaggcataggacccgtgtct</entry><entry>39</entry></row><row><entry>GAPD</entry><entry>LE</entry><entry>gggtgctaagcagttggtggtTTTTTaggcataggacccgtgtct</entry><entry>40</entry></row><row><entry>GAPD</entry><entry>LE</entry><entry>ccttccacgataccaaagttgtTTTTTaggcataggacccgtgtct</entry><entry>41</entry></row><row><entry>GAPD</entry><entry>LE</entry><entry>ggcatggactgtggtcatgagtTTTTTaggcataggacccgtgtct</entry><entry>42</entry></row><row><entry>GAPD</entry><entry>LE</entry><entry>agtcttctgggtggcagtgatTTTTTaggcataggacccgtgtct</entry><entry>43</entry></row><row><entry>GAPD</entry><entry>LE</entry><entry>ccatcacgccacagtttccTTTTTaggcataggacccgtgtct</entry><entry>44</entry></row><row><entry>GAPD</entry><entry>LE</entry><entry>cagtagaggcagggatgatgttcTTTTTaggcataggacccgtgtct</entry><entry>45</entry></row><row><entry>GAPD</entry><entry>LE</entry><entry>cacagccttggcagcgcTTTTTaggcataggacccgtgtct</entry><entry>46</entry></row><row><entry>GAPD</entry><entry>LE</entry><entry>ccagtgagcttcccgttcaTTTTTaggcataggacccgtgtct</entry><entry>47</entry></row><row><entry>GAPD</entry><entry>CE</entry><entry>tgacggtgccatggaatttTTTTTaaaactatacgtgct</entry><entry>48</entry></row><row><entry>GAPD</entry><entry>CE</entry><entry>agcttcccgttctcagcctTTTTTaaaactatacgtgct</entry><entry>49</entry></row><row><entry>GAPD</entry><entry>CE</entry><entry>tctcgctcctggaagatggtTTTTTaaaactatacgtgct</entry><entry>50</entry></row><row><entry>GAPD</entry><entry>CE</entry><entry>gcaaatgagccccagccTTTTTaaaactatacgtgct</entry><entry>51</entry></row><row><entry>GAPD</entry><entry>CE</entry><entry>ccttttggctcccccctTTTTTaaaactatacgtgct .</entry><entry>52</entry></row><row><entry>GAPD</entry><entry>CE</entry><entry>catggatgaccttggccagTTTTTaaaactatacgtgct</entry><entry>53</entry></row><row><entry>GAPD</entry><entry>CE</entry><entry>gctcagggatgaccttgccTTTTTaaaactatacgtgct</entry><entry>54</entry></row><row><entry>GAPD</entry><entry>CP</entry><entry>TTTTTTTTAGCACGTATAGTTTT</entry><entry>55</entry></row><row><entry>IFNG</entry><entry>BP</entry><entry>TGCATTAAAATATTTCTTAAGGTTTTCT</entry><entry>56</entry></row><row><entry>IFNG</entry><entry>BP</entry><entry>AAATGCCTAAGAAAAGAGTTCCA</entry><entry>57</entry></row><row><entry>IFNG</entry><entry>BP</entry><entry>AAAAAGTTTGAAGTAAAAGGAGACAAT</entry><entry>58</entry></row><row><entry>IFNG</entry><entry>BP</entry><entry>GATGCTCTGGTCATCTTTAAAGTTTTT</entry><entry>59</entry></row><row><entry>IFNG</entry><entry>BP</entry><entry>GGATGCTCTTCGACCTTGAAAC</entry><entry>60</entry></row><row><entry>IFNG</entry><entry>BP</entry><entry>AATAAATAGATTTAGATTTAAAATTCAAATATT</entry><entry>61</entry></row><row><entry>IFNG</entry><entry>LE</entry><entry>gcttcttttacatatgggtcctggTTTTTaggcataggacccgtgtct</entry><entry>62</entry></row><row><entry>IFNG</entry><entry>LE</entry><entry>ttatccgctacatctgaatgaccTTTTTaggcataggacccgtgtct</entry><entry>63</entry></row><row><entry>IFNG</entry><entry>LE</entry><entry>ttgatggtctccacactcttttgTTTTTaggcataggacccgtgtct</entry><entry>64</entry></row><row><entry>IFNG</entry><entry>LE</entry><entry>aaaaacttgacattcatgtcttccTTTTTaggcataggacccgtgtct</entry><entry>65</entry></row><row><entry>IFNG</entry><entry>LE</entry><entry>ataattagtcagcttttcgaagtcaTTTTTaggcataggacccgtgtct</entry><entry>66</entry></row><row><entry>IFNG</entry><entry>LE</entry><entry>tggacattcaagtcagttaccgaTTTTTaggcataggacccgtgtct</entry><entry>67</entry></row><row><entry>IFNG</entry><entry>LE</entry><entry>cgacagttcagccatcacttggTTTTTaggcataggacccgtgtct</entry><entry>68</entry></row><row><entry>IFNG</entry><entry>LE</entry><entry>agcatctgactcctttttcgcTTTTTaggcataggacccgtgtct</entry><entry>69</entry></row><row><entry>IFNG</entry><entry>LE</entry><entry>gcaggcaggacaaccattactgTTTTTaggcataggacccgtgtct</entry><entry>70</entry></row><row><entry>IFNG</entry><entry>LE</entry><entry>aatacttatttgattgatgagtctaaaaatTTTTTaggcataggacccgtgtct</entry><entry>71</entry></row><row><entry>IFNG</entry><entry>CE</entry><entry>cactctcctctttccaattcttcaTTTTTTTttcacacacattaac</entry><entry>72</entry></row><row><entry>IFNG</entry><entry>CE</entry><entry>ttggctctgcattatttttctgtTTTTTttcacacacattaac</entry><entry>73</entry></row><row><entry>IFNG</entry><entry>CE</entry><entry>tctcgtttctttttgttgctattgTTTTTttcacacacattaac</entry><entry>74</entry></row><row><entry>IFNG</entry><entry>CE</entry><entry>atgagttcatgtattgctttgcgtTTTTTttcacacacattaac</entry><entry>75</entry></row><row><entry>IFNG</entry><entry>CE</entry><entry>ttccctgttttagctgctggTTTTTttcacacacattaac</entry><entry>76</entry></row><row><entry>IFNG</entry><entry>CE</entry><entry>atattccccatataaataatgttaaatattTTTTTttcacacacattaac</entry><entry>77</entry></row><row><entry>IFNG</entry><entry>CP</entry><entry>TTTTTTTTGTTAATGTGTGTGAA</entry><entry>78</entry></row><row><entry>IL1</entry><entry>BP</entry><entry>GGTAAAACTGGATCATCTCAGACAA</entry><entry>79</entry></row><row><entry>IL1</entry><entry>BP</entry><entry>GGGGCATCACCTCCTCCA</entry><entry>80</entry></row><row><entry>IL1</entry><entry>BP</entry><entry>GTTCACATGCGCCTTGATGT</entry><entry>81</entry></row><row><entry>IL1</entry><entry>BP</entry><entry>GCTCTTGTTTTCACAGGGAAGA</entry><entry>82</entry></row><row><entry>IL1</entry><entry>BP</entry><entry>GGCTTTGTAGATGCCTTTCTCT</entry><entry>83</entry></row><row><entry>IL1</entry><entry>BP</entry><entry>gactgggtgccctggcc</entry><entry>84</entry></row><row><entry>IL1</entry><entry>LE</entry><entry>TaggcaggttgcctgggaTTTTTaggcataggacccgtgtct</entry><entry>85</entry></row><row><entry>IL1</entry><entry>LE</entry><entry>gtcttcactctgctgaaggcatTTTTTaggcataggacccgtgtct</entry><entry>86</entry></row><row><entry>IL1</entry><entry>LE</entry><entry>actcctttaacaacaagttgtccaTTTTTaggcataggacccgtgtct</entry><entry>87</entry></row><row><entry>IL1</entry><entry>LE</entry><entry>ccttaaagtcctccagcaaggTTTTTaggcataggacccgtgtct</entry><entry>88</entry></row><row><entry>IL1</entry><entry>LE</entry><entry>ggcttggcaacccaggtaacTTTTTaggcataggacccgtgtct</entry><entry>89</entry></row><row><entry>IL1</entry><entry>LE</entry><entry>caggttctcccccagggaTTTTTaggcataggacccgtgtct</entry><entry>90</entry></row><row><entry>IL1</entry><entry>LE</entry><entry>aatcgatgacagcgccgtaTTTTTaggcataggacccgtgtct</entry><entry>91</entry></row><row><entry>IL1</entry><entry>LE</entry><entry>cacctgctccacggccttTTTTTaggcataggacccgtgtct</entry><entry>92</entry></row><row><entry>IL1</entry><entry>LE</entry><entry>tggagcttattaaaggcattcttTTTTTaggcataggacccgtgtct</entry><entry>93</entry></row><row><entry>IL1</entry><entry>LE</entry><entry>tgatgaagatgtcaaactcactcatTTTTTaggcataggacccgtgtct</entry><entry>94</entry></row><row><entry>IL1</entry><entry>LE</entry><entry>cattgtcatgtaggcttctatgtagtTTTTTaggcataggacccgtgtct</entry><entry>95</entry></row><row><entry>IL1</entry><entry>LE</entry><entry>ccctgatgtctcagtttcgtatcttTTTTTaggcataggacccgtgtct</entry><entry>96</entry></row><row><entry>IL1</entry><entry>LE</entry><entry>tgtcctagagtctatagagtcgccaTTTTTaggcataggacccgtgtct</entry><entry>97</entry></row><row><entry>IL1</entry><entry>LE</entry><entry>gctatcccagagccccagatTTTTTaggcataggacccgtgtct</entry><entry>98</entry></row><row><entry>IL1</entry><entry>CE</entry><entry>agtgggtgcagctgttctcaTTTTTccgtgcttttctaat</entry><entry>99</entry></row><row><entry>IL1</entry><entry>CE</entry><entry>ctcggagatctcgaagcatgtTTTTTccgtgcttttctaat</entry><entry>100</entry></row><row><entry>IL1</entry><entry>CE</entry><entry>gctgatccttcatttgaaagaaaTTTTTccgtgcttttctaat</entry><entry>101</entry></row><row><entry>IL1</entry><entry>CE</entry><entry>ctgggtcttggttctcagcttTTTTTccgtgcttttctaat</entry><entry>102</entry></row><row><entry>IL1</entry><entry>CE</entry><entry>gcctcagcctgagggtcttTTTTTccgtgcttttctaat</entry><entry>103</entry></row><row><entry>IL1</entry><entry>CE</entry><entry>ccgattttggagacctctaatttaTTTTTccgtgcttttctaat</entry><entry>104</entry></row><row><entry>IL1</entry><entry>CP</entry><entry>TTTTTTTTATTAGAAAAGCACGG</entry><entry>105</entry></row><row><entry>IL1B</entry><entry>BP</entry><entry>ACTGACGCGGCCTGCC</entry><entry>106</entry></row><row><entry>IL1B</entry><entry>BP</entry><entry>ccagacatcaccaagctttttt</entry><entry>107</entry></row><row><entry>IL1B</entry><entry>LE</entry><entry>gccatcagcttcaaagaacaagTTTTTaggcataggacccgtgtct</entry><entry>108</entry></row><row><entry>IL1B</entry><entry>LE</entry><entry>aaggagcacttcatctgtttaggTTTTTaggcataggacccgtgtct</entry><entry>109</entry></row><row><entry>IL1B</entry><entry>LE</entry><entry>atgccgccatccagaggTTTTTaggcataggacccgtgtct</entry><entry>110</entry></row><row><entry>IL1B</entry><entry>LE</entry><entry>ggtcggagattcgtagctggTTTTTaggcataggacccgtgtct</entry><entry>111</entry></row><row><entry>IL1B</entry><entry>LE</entry><entry>gcttgtccatggccacaacaTTTTTaggcataggacccgtgtct</entry><entry>112</entry></row><row><entry>IL1B</entry><entry>LE</entry><entry>gggaaccagcatcttcctcaTTTTTaggcataggacccgtgtct</entry><entry>113</entry></row><row><entry>IL1B</entry><entry>LE</entry><entry>ggttcttcttcaaagatgaagggTTTTTaggcataggacccgtgtct</entry><entry>114</entry></row><row><entry>IL1B</entry><entry>LE</entry><entry>ttatcccatgtgtcgaagaagataTTTTTaggcataggacccgtgtct</entry><entry>115</entry></row><row><entry>IL1B</entry><entry>LE</entry><entry>catcgtgcacataagcctcgTTTTTaggcataggacccgtgtct</entry><entry>116</entry></row><row><entry>IL1B</entry><entry>LE</entry><entry>gcagttcagtgatcgtacaggtgTTTTTaggcataggacccgtgtct</entry><entry>117</entry></row><row><entry>IL1B</entry><entry>LE</entry><entry>gctgtgagtcccggagcgtTTTTTaggcataggacccgtgtct</entry><entry>118</entry></row><row><entry>IL1B</entry><entry>LE</entry><entry>atggagaacaccacttgttgctTTTTTaggcataggacccgtgtct</entry><entry>119</entry></row><row><entry>IL1B</entry><entry>LE</entry><entry>actttcttctccttgtacaaaggacTTTTTaggcataggacccgtgtct</entry><entry>120</entry></row><row><entry>IL1B</entry><entry>LE</entry><entry>aggccacaggtattttgtcattTTTTTaggcataggacccgtgtct</entry><entry>121</entry></row><row><entry>IL1B</entry><entry>CE</entry><entry>gcagaggtccaggtcctggTTTTTaacgtgtattccatt</entry><entry>122</entry></row><row><entry>IL1B</entry><entry>CE</entry><entry>tgaagcccttgctgtagtggtTTTTTaacgtgtattccatt</entry><entry>123</entry></row><row><entry>IL1B</entry><entry>CE</entry><entry>cctggaaggtctgtgggcaTTTTTaacgtgtattccatt</entry><entry>124</entry></row><row><entry>IL1B</entry><entry>CE</entry><entry>aaagaaggtgctcaggtcattctTTTTTaacgtgtattccatt</entry><entry>125</entry></row><row><entry>IL1B</entry><entry>CE</entry><entry>ggagagctttcagttcatatggaTTTTTaacgtgtattccatt</entry><entry>126</entry></row><row><entry>IL1B</entry><entry>CE</entry><entry>ccatatcctgtccctggaggtTTTTTaacgtgtattccatt</entry><entry>127</entry></row><row><entry>IL1B</entry><entry>CE</entry><entry>attcttttccttgaggcccaTTTTTaacgtgtattccatt</entry><entry>128</entry></row><row><entry>IL1B</entry><entry>CP</entry><entry>TTTTTTTTAATGGAATACACGTT</entry><entry>129</entry></row><row><entry>IL2</entry><entry>BP</entry><entry>TTCCATTCAAAATCATCTGTAAATC</entry><entry>130</entry></row><row><entry>IL2</entry><entry>BP</entry><entry>CCTGGGTCTTAAGTGAAAGTTTTT</entry><entry>131</entry></row><row><entry>IL2</entry><entry>BP</entry><entry>AGCATATTCACACATGAATGTTGTT</entry><entry>132</entry></row><row><entry>IL2</entry><entry>BP</entry><entry>AAAAGGTAATCCATCTGTTCAGAAA</entry><entry>133</entry></row><row><entry>IL2</entry><entry>BP</entry><entry>ATTCAACAATAAATATAAAATTTAAATATfiTA</entry><entry>134</entry></row><row><entry>IL2</entry><entry>LE</entry><entry>agtaggtgcactgtttgtgacaagTTTTTaggcataggacccgtgtct</entry><entry>135</entry></row><row><entry>IL2</entry><entry>LE</entry><entry>gctgtgttttctttgtagaacttgaTTTTTaggcataggacccgtgtct</entry><entry>136</entry></row><row><entry>IL2</entry><entry>LE</entry><entry>cagcagtaaatgctccagttgtaTTTTTaggcataggacccgtgtct</entry><entry>137</entry></row><row><entry>IL2</entry><entry>LE</entry><entry>aaacttaaatgtgagcatcctggTTTTTaggcataggacccgtgtct</entry><entry>138</entry></row><row><entry>IL2</entry><entry>LE</entry><entry>tagacactgaagatgtttcagttctgTTTTTaggcataggacccgtgtct</entry><entry>139</entry></row><row><entry>IL2</entry><entry>LE</entry><entry>gctttgagctaaatttagcacttcTTTTTaggcataggacccgtgtct</entry><entry>140</entry></row><row><entry>IL2</entry><entry>LE</entry><entry>attacgttgatattgctgattaagtcTTTTTaggcataggacccgtgtct</entry><entry>141</entry></row><row><entry>IL2</entry><entry>LE</entry><entry>ttctacaatggttgctgtctcatcTTTTTaggcataggacccgtgtct</entry><entry>142</entry></row><row><entry>IL2</entry><entry>LE</entry><entry>tcagtgttgagatgatgctttgacTTTTfiaggcataggacccgtgtct</entry><entry>143</entry></row><row><entry>IL2</entry><entry>LE</entry><entry>agtgggaagcacttaattatcaagTTTTTaggcataggacccgtgtct</entry><entry>144</entry></row><row><entry>IL2</entry><entry>LE</entry><entry>aatagttacaataggtagcaaaccatacTTTTTaggcataggacccgtgtct</entry><entry>145</entry></row><row><entry>IL2</entry><entry>CE</entry><entry>tgagtttgggattcttgtaattattaaTTTTTgaagttaccgttttc</entry><entry>146</entry></row><row><entry>IL2</entry><entry>CE</entry><entry>tggccttcttgggcatgtaTTTTTgaagttaccgttttc</entry><entry>147</entry></row><row><entry>IL2</entry><entry>CE</entry><entry>ctccagaggtttgagttcttcttcTTTTTgaagttaccgttttc</entry><entry>148</entry></row><row><entry>IL2</entry><entry>CE</entry><entry>tcagatccctttagttccagaactTTTTTgaagttaccgttttc</entry><entry>149</entry></row><row><entry>IL2</entry><entry>CE</entry><entry>aataaatagaaggcctgatatgttttaTTTTTgaagttaccgttttc</entry><entry>150</entry></row><row><entry>IL2</entry><entry>CP</entry><entry>TTTTTTTTGAAAACGGTAACTTC</entry><entry>151</entry></row><row><entry>IL6</entry><entry>BP</entry><entry>TGGGGCAGGGAAGGCA</entry><entry>152</entry></row><row><entry>IL6</entry><entry>BP</entry><entry>GGAATCTTCTCCTGGGGGTAC</entry><entry>153</entry></row><row><entry>IL6</entry><entry>BP</entry><entry>TGGGGCGGCTACATCTTT</entry><entry>154</entry></row><row><entry>IL6</entry><entry>BP</entry><entry>GCTTTCACACATGTTACTCTTGTTACA</entry><entry>155</entry></row><row><entry>IL6</entry><entry>BP</entry><entry>TTTGGAAGGTTCAGGTTGTTTT</entry><entry>156</entry></row><row><entry>IL6</entry><entry>BP</entry><entry>CCTCAAACTCCAAAAGACCAGTG</entry><entry>157</entry></row><row><entry>IL6</entry><entry>BP</entry><entry>TTGGGTCAGGGGTGGTTATT</entry><entry>158</entry></row><row><entry>IL6</entry><entry>BP</entry><entry>CTGCAGGAACTCCTTAAAGCTG</entry><entry>159</entry></row><row><entry>IL6</entry><entry>BP</entry><entry>CCCATTAACAACAACAATCTGAGG</entry><entry>160</entry></row><row><entry>IL26</entry><entry>LE</entry><entry>ggctcctggaggcgagataTTTTTaggcataggacccgtgtct</entry><entry>161</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>aactggaccgaaggcgctTTTTTaggcataggacccgtgtct</entry><entry>162</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>gcaggcaacaccaggagcTTTTTaggcataggacccgtgtct</entry><entry>163</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>aagaggtgagtggctgtctgtgTTTTTaggcataggacccgtgtct</entry><entry>164</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>gaatttgtttgtcaattcgttctgTTTTTaggcataggacccgtgtct</entry><entry>165</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>gatgccgtcgaggatgtaccTTTTTaggcataggacccgtgtct</entry><entry>166</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>ctgccagtgcctctttgctTTTTTaggcataggacccgtgtct</entry><entry>167</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>gcatccatctttttcagccatcTTTTTaggcataggacccgtgtct</entry><entry>168</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>atgattttcaccaggcaagtctTTTTTaggcataggacccgtgtct</entry><entry>169</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>atctgttctggaggtactctaggtataTTTTTaggcataggacccgtgtct</entry><entry>170</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>ggcttgttcctcactactctcaaTTTTTaggcataggacccgtgtct</entry><entry>171</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>ttttgtactcatctgcacagctctTTTTTaggcataggacccgtgtct</entry><entry>172</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>ctgcaggaactggatcaggacTTTTTaggcataggacccgtgtct</entry><entry>173</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>gcatctagattctttgcctttttTTTTTaggcataggacccgtgtct</entry><entry>174</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>gcaggctggcatttgtggTTTTTaggcataggacccgtgtct</entry><entry>175</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>tgtgcctgcagcttcgtcaTTTTTaggcataggacccgtgtct</entry><entry>176</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>tgtcctgcagccactggttcTTTTTaggcataggacccgtgtct</entry><entry>177</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>cgcagaatgagatgagttgtcaTTTTTaggcataggacccgtgtct</entry><entry>178</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>tgcccatgctacatttgccTTTTTaggcataggacccgtgtct</entry><entry>179</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>ggtttctgaccagaagaaggaatgTTTTTaggcataggacccgtgtct</entry><entry>180</entry></row><row><entry>IL6</entry><entry>LE</entry><entry>aagttctgtgcccagtggacaTTTTTaggcataggacccgtgtct</entry><entry>181</entry></row><row><entry>IL6</entry><entry>CE</entry><entry>gagcttctctttcgttcccgTTTTTggggaacatagaaaa</entry><entry>182</entry></row><row><entry>IL6</entry><entry>CE</entry><entry>tgtggagaaggagttcatagctgTTTTTggggaacatagaaaa</entry><entry>183</entry></row><row><entry>IL6</entry><entry>CE</entry><entry>agccccagggagaaggcTTTTTggggaacatagaaaa</entry><entry>184</entry></row><row><entry>IL6</entry><entry>CE</entry><entry>tgtctcctttctcagggctgaTTTTTggggaacatagaaaa</entry><entry>185</entry></row><row><entry>IL6</entry><entry>CE</entry><entry>cctcattgaatccagattggaaTTTTTggggaacatagaaaa</entry><entry>186</entry></row><row><entry>IL6</entry><entry>CE</entry><entry>gaagagccctcaggctggaTTTTTggggaacatagaaaa</entry><entry>187</entry></row><row><entry>IL6</entry><entry>CP</entry><entry>TTTTTTTTTTTTCTATGTTCCCC</entry><entry>188</entry></row><row><entry>IL8</entry><entry>BP</entry><entry>CAAAAACTTCTCCACAACCCTC</entry><entry>189</entry></row><row><entry>IL8</entry><entry>BP</entry><entry>AGTGTTGAAGTAGATTTGCTTGAAGT</entry><entry>190</entry></row><row><entry>IL8</entry><entry>BP</entry><entry>CAACAGACCCACACAATACATGA</entry><entry>191</entry></row><row><entry>IL8</entry><entry>BP</entry><entry>GTACAATGAAAAACTATTCATTGTTTACT</entry><entry>192</entry></row><row><entry>IL8</entry><entry>BP</entry><entry>TTTTTTGTAGATTCAAATAAATAATACTTTA</entry><entry>193</entry></row><row><entry>IL8</entry><entry>BP</entry><entry>AAATCCTTATATTTAAAAATTATTTGTTG</entry><entry>194</entry></row><row><entry>IL8</entry><entry>BP</entry><entry>GCTTCAAATATCACATTCTAGCAAAC</entry><entry>195</entry></row><row><entry>IL8</entry><entry>BP</entry><entry>AAAAAATCCAGGATTTCCAGCt</entry><entry>196</entry></row><row><entry>IL8</entry><entry>BP</entry><entry>CTAGGGTTGCCAGATTTAACAGA</entry><entry>197</entry></row><row><entry>IL8</entry><entry>BP</entry><entry>CCACTTAGAAATAAAGGAGAAACCA</entry><entry>198</entry></row><row><entry>IL8</entry><entry>BP</entry><entry>CATGTCCTCACAACATCACTGTGA</entry><entry>199</entry></row><row><entry>IL8</entry><entry>BP</entry><entry>ATGAAAAAACTTAAAGTGCTTCCA</entry><entry>200</entry></row><row><entry>IL8</entry><entry>BP</entry><entry>AAGTTACACTTGAAAATAATTTATGTTATG</entry><entry>201</entry></row><row><entry>IL8</entry><entry>BP</entry><entry>TTAAATAAATACATAAATAATAA.ATAGGTTAAT</entry><entry>202</entry></row><row><entry>IL8</entry><entry>BP</entry><entry>ATAAAACATCATTTAATATCTAAAATAAAAT</entry><entry>203</entry></row><row><entry>IL8</entry><entry>BP</entry><entry>TAAAAACCCTGATTGAAATTTATCTA</entry><entry>204</entry></row><row><entry>IL8</entry><entry>LE</entry><entry>ggtccagacagagctctcttccTTTTTaggcataggacccgtgtct</entry><entry>205</entry></row><row><entry>IL8</entry><entry>LE</entry><entry>ttggataccacagagaatgaatttTTTTTaggcataggacccgtgtct</entry><entry>206</entry></row><row><entry>IL8</entry><entry>LE</entry><entry>ttcactggcatcttcactgattcTTTTTaggcataggacccgtgtct</entry><entry>207</entry></row><row><entry>IL8</entry><entry>LE</entry><entry>tgtattgcatctggcaaccctaTTTTTaggcataggacccgtgtct</entry><entry>208</entry></row><row><entry>IL8</entry><entry>LE</entry><entry>gaaattcaaatttaaccaggaatctTTTTTaggcataggacccgtgtct</entry><entry>209</entry></row><row><entry>IL8</entry><entry>LE</entry><entry>catataagtatgttctggatatttcatgTTTTTaggcataggacccgtgtct</entry><entry>210</entry></row><row><entry>IL8</entry><entry>LE</entry><entry>ttctcccgtgcaatatctaggaTTTTTaggcataggacccgtgtct</entry><entry>211</entry></row><row><entry>IL8</entry><entry>LE</entry><entry>ggcctcaattttgctatttgtataTTTTTaggcataggacccgtgtct</entry><entry>212</entry></row><row><entry>IL8</entry><entry>LE</entry><entry>ccattcaattcctgaaattaaagttTTTTTaggcataggacccgtgtct</entry><entry>213</entry></row><row><entry>IL8</entry><entry>LE</entry><entry>attgtcccatcatttttatgtgatTTTTTaggcataggacccgtgtct</entry><entry>214</entry></row><row><entry>IL8</entry><entry>LE</entry><entry>aaatttgactttatggcaaaatttTTTTTaggcataggacccgtgtct</entry><entry>215</entry></row><row><entry>IL8</entry><entry>LE</entry><entry>aggcacagtggaacaaggactTTTTTaggcataggacccgtgtct</entry><entry>216</entry></row><row><entry>IL8</entry><entry>LE</entry><entry>ggtaagatggtggctaatactttttTTTTTaggcataggacccgtgtct</entry><entry>217</entry></row><row><entry>IL8</entry><entry>LE</entry><entry>aattcttgcacaaatatttgatgcTTTTTaggcataggacccgtgtct</entry><entry>218</entry></row><row><entry>IL8</entry><entry>LE</entry><entry>caatgattcatcttctatttttccaTTTTTaggcataggacccgtgtct</entry><entry>219</entry></row><row><entry>IL8</entry><entry>LE</entry><entry><img file="EP1880025B1_D0001.tif" /></entry><entry>220</entry></row><row><entry>IL8</entry><entry>CE</entry><entry>tgcacccagttttccttggTTTTTttcaaatgttagcct</entry><entry>221</entry></row><row><entry>IL8</entry><entry>CE</entry><entry>ttttatgaattctcagccctcttTTTTTttcaaatgttagcct</entry><entry>222</entry></row><row><entry>IL8</entry><entry>CE</entry><entry>cggatattctcttggcccttTTTTTttcaaatgttagcct</entry><entry>223</entry></row><row><entry>IL8</entry><entry>CE</entry><entry>tgtggatcctggctagcagaTTTTTttcaaatgttagcct</entry><entry>224</entry></row><row><entry>IL8</entry><entry>CE</entry><entry>acccaattgtttgtttgtttaatcTTTTTttcaaatgttagcct</entry><entry>225</entry></row><row><entry>IL8</entry><entry>CP</entry><entry>TTTTTTTTAGGCTAACATTTGAA</entry><entry>226</entry></row><row><entry>TNF</entry><entry>BP</entry><entry>CCCTCTGGGGGCCGA</entry><entry>227</entry></row><row><entry>TNF</entry><entry>BP</entry><entry>GAGGTCCCTGGGGAACTCTT</entry><entry>228</entry></row><row><entry>TNF</entry><entry>BP</entry><entry>ggccagagggctgattagaga</entry><entry>229</entry></row><row><entry>TNF</entry><entry>BP</entry><entry>AGGCTTGTCACTCGGGGTT</entry><entry>230</entry></row><row><entry>TNF</entry><entry>BP</entry><entry>tgaagaggacctgggagtagatg</entry><entry>231</entry></row><row><entry>TNF</entry><entry>BP</entry><entry>GGGCAGCCTTGGCCCT</entry><entry>232</entry></row><row><entry>TNF</entry><entry>BP</entry><entry>TGGCAGGGGCTCTTGATG</entry><entry>233</entry></row><row><entry>TNF</entry><entry>BP</entry><entry>CCCCTCTGGGGTCTCCCTC</entry><entry>234</entry></row><row><entry>TNF</entry><entry>BP</entry><entry>GTTTGGGAAGGTTGGATGTTC</entry><entry>235</entry></row><row><entry>TNF</entry><entry>BP</entry><entry>TGGGGCAGGGGAGGC</entry><entry>236</entry></row><row><entry>TNF</entry><entry>BP</entry><entry>AGGAGGGGGTAATAAAGGGAT</entry><entry>237</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>tcactccaaagtgcagcaggTTTTTaggcataggacccgtgtct</entry><entry>238</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>ggtttgctacaacatgggctacTTTTTaggcataggacccgtgtct</entry><entry>239</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>ggcggttcagccactggaTTTTTaggcataggacccgtgtct</entry><entry>240</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>caggagggcattggcccTTTTTaggcataggacccgtgtct</entry><entry>241</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>agctccacgccattggcTTTTTaggcataggacccgtgtct</entry><entry>242</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>caccaccagctggttatctctcTTTTTaggcataggacccgtgtct</entry><entry>243</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>aggtacaggccctctgatggTTTTTaggcataggacccgtgtct</entry><entry>244</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>tgaggagcacatgggtggagTTTTTaggcataggacccgtgtct</entry><entry>245</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>gcggctgatggtgtgggTTTTTaggcataggacccgtgtct</entry><entry>246</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>gcagagaggaggttgaccttgTTTTTaggcataggacccgtgtct</entry><entry>247</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>cagggcttggcctcagcTTTTTaggcataggacccgtgtct</entry><entry>248</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>tctccagctggaagaccccTTTTTaggcataggacccgtgtct</entry><entry>249</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>gcgctgagtcggtcaccctTTTTTaggcataggacccgtgtct</entry><entry>250</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>agactcggcaaagtcgagatagTTTTTaggcataggacccgtgtct</entry><entry>251</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>atcccaaagtagacctgcccTTTTTaggcataggacccgtgtct</entry><entry>252</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>gtcctcctcacagggcaatgTTTTTaggcataggacccgtgtct</entry><entry>253</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>cagaagaggttgagggtgtctgaTTTTTaggcataggacccgtgtct</entry><entry>254</entry></row><row><entry>TNF</entry><entry>LE</entry><entry>gcttgggttccgaccctaagTTTTTaggcataggacccgtgtct</entry><entry>255</entry></row><row><entry>TNF</entry><entry>CE</entry><entry>cgagaagatgatctgactgcctgTTTTTctgagtcaaagcatt</entry><entry>256</entry></row><row><entry>TNF</entry><entry>CE</entry><entry>gctgcccctcagcttgagTTTTTctgagtcaaagcatt</entry><entry>257</entry></row><row><entry>TNF</entry><entry>CE</entry><entry>gtctggtaggagacggcgatTTTTTctgagtcaaagcatt</entry><entry>258</entry></row><row><entry>TNF</entry><entry>CE</entry><entry>tcccagatagatgggctcatacTTTTTctgagtcaaagcatt</entry><entry>259</entry></row><row><entry>TNF</entry><entry>CE</entry><entry>tcgggccgattgatctcaTTTTTctgagtcaaagcatt</entry><entry>260</entry></row><row><entry>TNF</entry><entry>CE</entry><entry>cccccaattctctttttgagcTTTTTctgagtcaaagcatt</entry><entry>261</entry></row><row><entry>TNF</entry><entry>CP</entry><entry>TTTTTTTTAATGCTTTGACTCAG</entry><entry>262</entry></row><row><entry>VEGF</entry><entry>BP</entry><entry>CATCAGGGGCACACAGGATG</entry><entry>263</entry></row><row><entry>VEGF</entry><entry>BP</entry><entry>GCAGCCCCCGCATCG</entry><entry>264</entry></row><row><entry>VEGF</entry><entry>BP</entry><entry>CTCCTCAGTGGGCACACACTC</entry><entry>265</entry></row><row><entry>VEGF</entry><entry>LE</entry><entry>tggaggtagagcagcaaggcTTTTTaggcataggacccgtgtct</entry><entry>266</entry></row><row><entry>VEGF</entry><entry>LE</entry><entry>tgggaccacttggcatggTTTTTaggcataggacccgtgtct</entry><entry>267</entry></row><row><entry>VEGF</entry><entry>LE</entry><entry>gatgattctgccctcctccttTTTTTaggcataggacccgtgtct</entry><entry>268</entry></row><row><entry>VEGF</entry><entry>LE</entry><entry>tccatgaacttcaccacttcgtTTTTTaggcataggacccgtgtct</entry><entry>269</entry></row><row><entry>VEGF</entry><entry>LE</entry><entry>gcagtagctgcgctgatagacaTTTTTaggcataggacccgtgtct</entry><entry>270</entry></row><row><entry>VEGF</entry><entry>LE</entry><entry>accagggtctcgattggatgTTTTTaggcataggacccgtgtct</entry><entry>271</entry></row><row><entry>VEGF</entry><entry>LE</entry><entry>agggtactcctggaagatgtccTTTTTaggcataggacccgtgtct</entry><entry>272</entry></row><row><entry>VEGF</entry><entry>LE</entry><entry>gcttgaagatgtactcgatctcatcTTTTTaggcataggacccgtgtct</entry><entry>273</entry></row><row><entry>VEGF</entry><entry>LE</entry><entry>caggccctcgtcattgcaTTTTTaggcataggacccgtgtct</entry><entry>274</entry></row><row><entry>VEGF</entry><entry>LE</entry><entry>TaatctgcatggtgatgttggaTTTTTaggcataggacccgtgtct</entry><entry>275</entry></row><row><entry>VEGF</entry><entry>LE</entry><entry>atttgttgtgctgtaggaagctcTTTTTaggcataggacccgtgtct</entry><entry>276</entry></row><row><entry>VEGF</entry><entry>LE</entry><entry>ctgattttttttcttgtcttgctctTTTTTaggcataggacccgtgtct</entry><entry>277</entry></row><row><entry>VEGF</entry><entry>LE</entry><entry>ttgcgctttcgtttttgcTTTTTaggcataggacccgtgtct</entry><entry>278</entry></row><row><entry>VEGF</entry><entry>LE</entry><entry>ggcccacagggaacgctTTTTTaggcataggacccgtgtct</entry><entry>279</entry></row><row><entry>VEGF</entry><entry>CE</entry><entry>aaggctccaatgcacccaTTTTTctttgagttcggttt</entry><entry>280</entry></row><row><entry>VEGF</entry><entry>CE</entry><entry>ctgccatgggtgcagccTTTTTctttgagttcggttt</entry><entry>281</entry></row><row><entry>VEGF</entry><entry>CE</entry><entry>tggtgaggtttgatccgcaTTTTTctttgagttcggttt</entry><entry>282</entry></row><row><entry>VEGF</entry><entry>CE</entry><entry>atctctcctatgtgctggcctTTTTTctttgagttcggttt</entry><entry>283</entry></row><row><entry>VEGF</entry><entry>CE</entry><entry>atctttctttggtctgcattcacTTTTTctttgagttcggttt</entry><entry>284</entry></row><row><entry>VEGF</entry><entry>CE</entry><entry>ccctttccctttcctcgaaTTTTTctttgagttcggttt</entry><entry>285</entry></row><row><entry>VEGF</entry><entry>CE</entry><entry>ccaggacttataccgggatttcTTTTTctttgagttcggttt</entry><entry>286</entry></row><row><entry>VEGF</entry><entry>CP</entry><entry>TTTTTTTTAAACCGAACTCAAAG</entry><entry>287</entry></row></tbody></tgroup></table></tables>

Regulation of gene expression from cellular models of inflammation and apoptosis

0182To demonstrate the utility of this assay using cell lysates, two well-characterized cell culture model systems that elicit expression changes in genes were employed within the panels. The first model system utilized PMA/LPS treatment of U937 cells to monitor the changes of cytokine gene expression in inflammatory responses, and the second model utilized TNFα-treated HeLa cells to monitor expression changes in pro- and anti-apoptotic genes.

0183U937 cells with or without PMA/LPS treatment were lysed in the culture flask by adding a lysis buffer (from QuantiGene and QuantiGene Plex kit) directly to the culture media. Since the lysis buffer facilitates the release and hybridization of intact mRNA, crude cell lysates were used directly for all multiplex bDNA assays. Gene expression was measured in the 10-plex cytokine panel for control and PMA/LPS treated U937 cells. 40,000 cell equivalents of cell lysate was used in the assay. When the assay signal for a gene was below the detection limit, the expression level of that gene was not plotted. The measurements were performed in quadruplicate and data acquired from Luminex 100™ is normalized to the housekeeping gene GAPD. Error bar represents 1 s.d.

0184As shown in <figref idref="f0011"><b>Figure 8</b></figref><b>Panel A,</b> untreated U937 cells, GAPDH and VEGF were expressed at a high level, IL8, IL1b, and TNFα were expressed at moderate to low level, and IL6, IL10, CSF2 (GM-CSF), IL2, and IFN-γ are not significantly above the background. PMA/LPS treatment strongly induced the expression of proinflammatory cytokines IL8, IL-1b and TNFα. However, cytokines IL6, IL10 and CSF2 were only moderately induced, while IFN-γ and IL2 and VEGF remain essentially unchanged. The average %CV or the measurements (including both signal and background) for uninduced cells was 12.8%, and for induced cells was 8.9%. The signals of the induced genes increased linearly with the increasing amount of cell lysate (e.g., 6,000 to 25,000 cells) assayed, while the background level remained the same throughout, demonstrating the specific nature of the assay signal. Further verification of mRNA levels from the same cell lysates was performed using the well-established single-plex bDNA assay (<figref idref="f0011"><b>Figure 8</b></figref><b>Panel B</b>) . 20,000 cell equivalents of cell lysate was used in the assay. The measurements were performed in quadruplicate and data normalized to 40,000 cell equivalents and to housekeeping gene GAPD. Error bar represents 1 s.d. The results showed a highly similar pattern of gene expression to the results of the multiplex assay, with a correlation factor of 0.94 by matched pair analysis (<figref idref="f0011"><b>Figure 8</b></figref><b>Panel B</b>).

0185To further demonstrate the utility of the multiplex assay, two 10-plex apoptosis panels were developed and used to analyze gene expression from HeLa cells treated with TNFα for 1 to 6 hours. One apoptosis panel measures the expression of several major subunits of the NF-kB transcription complexes, including RelA, RelB, NFKB1, NFKB2, and NFKB1A, as well as a number of additional pro- and anti-apoptotic regulators, and a number of cytokines (<b>Table 1</b>). Expression of individual genes in the 10-plex apoptosis panel with or without TNFα treatment for 0, 1, 3, or 6 hours is shown in <figref idref="f0012 f0013"><b>Figure 9</b></figref><b>Panel A.</b> 80,000 cell equivalents of cell lysate was used in the assay except for GAPD where data was obtained from 10,000 cell equivalents of cell lysate. The measurements were performed in triplicate with data acquired from a Bio-Plex instrument at high sensitivity setting and normalized to housekeeping gene GAPD.

0186Several patterns of gene expression were observed (<figref idref="f0012 f0013"><b>Figure 9</b></figref><b>Panel A</b>). The expression of A20 and TNFα was rapidly elevated after 1 hour, then declined after 3 hour, and further declined after 6 hour of TNFα stimulation. The expression of NFKB1, NFKB2, NFKB1A, and IL-6 was increased after 1 hour, and maintained at roughly the similar level (within 2-fold of each other) after 3 hour and 6 hour of TNFα stimulation. The expression of RELB increased moderately at 1 hour, but elevated at much higher level after 3 hours and 6 hours of TNFα stimulation. Consistent with their known functional role in the NF-kB transcription complex and in cell cycle control, the expression levels of RELA and CDKN1A did not change significantly upon TNFα treatment. The average %CV of all expression measurements was 8.5%. Verification tests of these results were performed by measuring the expression levels of all ten genes in control and 3 hour TNFα-treated samples with the single-plex bDNA assay. 40,000 cell equivalents of cell lysate was used in each assay except for GAPD where data was obtained from 10,000 cell equivalents of cell lysate. Measurements from both technologies reveal similar relative expression levels and fold-of-induction changes (<figref idref="f0012 f0013"><b>Figure 9</b></figref><b>Panel B</b>). Correlation (R²) between expression measurements for untreated samples was >0.99, and for treated samples R² >0.94 was observed across both panels.

0187Performance evaluation data for the two apoptosis panels is summarized in <b>Tables 4</b> and <b>5</b>.

0188<tables id="tabl0004" num="0004"><table frame="all"><title><b>Table 4.</b> Detection sensitivity, cross-reactivity, and assay background of the apoptosis 1 panel.</title><tgroup cols="11"><colspec colnum="1" colname="col1" colwidth="43mm" /><colspec colnum="2" colname="col2" colwidth="17mm" /><colspec colnum="3" colname="col3" colwidth="17mm" /><colspec colnum="4" colname="col4" colwidth="17mm" /><colspec colnum="5" colname="col5" colwidth="16mm" /><colspec colnum="6" colname="col6" colwidth="17mm" /><colspec colnum="7" colname="col7" colwidth="14mm" /><colspec colnum="8" colname="col8" colwidth="18mm" /><colspec colnum="9" colname="col9" colwidth="18mm" /><colspec colnum="10" colname="col10" colwidth="17mm" /><colspec colnum="11" colname="col11" colwidth="18mm" /><thead><row><entry align="center" valign="top">Gene</entry><entry align="center" valign="top">RELA</entry><entry align="center" valign="top">NFKB2</entry><entry align="center" valign="top">NKFB1</entry><entry align="center" valign="top">RELB</entry><entry align="center" valign="top">IL6</entry><entry align="center" valign="top">GAPD</entry><entry align="center" valign="top">NFKB1A</entry><entry align="center" valign="top">TNFAIP3</entry><entry align="center" valign="top">TNFA</entry><entry align="center" valign="top">CDKN1A</entry></row></thead><tbody><row><entry align="center">LOD (amol)</entry><entry align="center" valign="middle">0.06</entry><entry align="center" valign="middle">0.06</entry><entry align="center" valign="middle">0.04</entry><entry align="center" valign="middle">0.04</entry><entry align="center" valign="middle">0.04</entry><entry align="center" valign="middle">0.04</entry><entry align="center" valign="middle">0.16</entry><entry align="center" valign="middle">0.04</entry><entry align="center" valign="middle">0.04</entry><entry align="center" valign="middle">0.04</entry></row><row><entry align="center">% Cross- reactivity</entry><entry align="center" valign="middle">0.024 %</entry><entry align="center" valign="middle">0.028 %</entry><entry align="center" valign="middle">0.023 %</entry><entry align="center" valign="middle">0.028%</entry><entry align="center" valign="middle">0.005 %</entry><entry align="center" valign="middle">0.02 %</entry><entry align="center" valign="middle">0.017 %</entry><entry align="center" valign="middle">0.019 %</entry><entry align="center" valign="middle">0.044 %</entry><entry align="center" valign="middle">0.023 %</entry></row><row><entry align="center">Average background (MFI)</entry><entry align="center" valign="middle">53.3</entry><entry align="center" valign="middle">19.5</entry><entry align="center" valign="middle">10.1</entry><entry align="center" valign="middle">14.5</entry><entry align="center" valign="middle">15.9</entry><entry align="center" valign="middle">18.1</entry><entry align="center" valign="middle">82.1</entry><entry align="center" valign="middle">31.1</entry><entry align="center" valign="middle">15.6</entry><entry align="center" valign="middle">16</entry></row><row><entry align="center">% Intra- plate CV</entry><entry align="center" valign="middle">13</entry><entry align="center" valign="middle">10</entry><entry align="center" valign="middle">14</entry><entry align="center" valign="middle">13</entry><entry align="center" valign="middle">9</entry><entry align="center" valign="middle">9</entry><entry align="center" valign="middle">12</entry><entry align="center" valign="middle">11</entry><entry align="center" valign="middle">11</entry><entry align="center" valign="middle">9</entry></row><row><entry align="center">% Inter- plate CV</entry><entry align="center" valign="middle">16</entry><entry align="center" valign="middle">16</entry><entry align="center" valign="middle">14</entry><entry align="center" valign="middle">13</entry><entry align="center" valign="middle">8</entry><entry align="center" valign="middle">12</entry><entry align="center" valign="middle">10</entry><entry align="center" valign="middle">14</entry><entry align="center" valign="middle">12</entry><entry align="center" valign="middle">13</entry></row></tbody></tgroup></table></tables>

0189<tables id="tabl0005" num="0005"><table frame="all"><title><b>Table 5.</b> Detection sensitivity, cross-reactivity, and assay background of the apoptosis 2 panel.</title><tgroup cols="11"><colspec colnum="1" colname="col1" colwidth="43mm" /><colspec colnum="2" colname="col2" colwidth="17mm" /><colspec colnum="3" colname="col3" colwidth="17mm" /><colspec colnum="4" colname="col4" colwidth="17mm" /><colspec colnum="5" colname="col5" colwidth="17mm" /><colspec colnum="6" colname="col6" colwidth="17mm" /><colspec colnum="7" colname="col7" colwidth="17mm" /><colspec colnum="8" colname="col8" colwidth="18mm" /><colspec colnum="9" colname="col9" colwidth="20mm" /><colspec colnum="10" colname="col10" colwidth="17mm" /><colspec colnum="11" colname="col11" colwidth="17mm" /><thead><row><entry align="center" valign="top">Gene</entry><entry align="center" valign="top">BCL2L1</entry><entry align="center" valign="top">IL6R</entry><entry align="center" valign="top">BCL2</entry><entry align="center" valign="top">BAK1</entry><entry align="center" valign="top">PTK2B</entry><entry align="center" valign="top">ACTB</entry><entry align="center" valign="top">CFLAR</entry><entry align="center" valign="top">TNFRSF6</entry><entry align="center" valign="top">TNFSF6</entry><entry align="center" valign="top">BAD</entry></row></thead><tbody><row><entry align="center">LOD (amol)</entry><entry align="center">0.04</entry><entry align="center">0.04</entry><entry align="center">0.16</entry><entry align="center">0.16</entry><entry align="center">0.04</entry><entry align="center">0.04</entry><entry align="center">0.06</entry><entry align="center">0.04</entry><entry align="center">0.04</entry><entry align="center">0.16</entry></row><row><entry align="center">% Cross-reactivity</entry><entry align="center">0.021 %</entry><entry align="center">0.005 %</entry><entry align="center">0.038 %</entry><entry align="center">0.072 %</entry><entry align="center">0.006 %</entry><entry align="center">0.016 %</entry><entry align="center">0.021 %</entry><entry align="center">0.038 %</entry><entry align="center">0.345 %</entry><entry align="center">0.024 %</entry></row><row><entry align="center">Average background (MFI)</entry><entry align="center" valign="middle">9.9</entry><entry align="center" valign="middle">24.3</entry><entry align="center" valign="middle">72.6</entry><entry align="center" valign="middle">26.4</entry><entry align="center" valign="middle">15.3</entry><entry align="center" valign="middle">11.6</entry><entry align="center" valign="middle">24.4</entry><entry align="center" valign="middle">16.5</entry><entry align="center" valign="middle">17.8</entry><entry align="center" valign="middle">14.8</entry></row><row><entry align="center">% Intra-plate CV</entry><entry align="center" valign="middle">7</entry><entry align="center" valign="middle">7</entry><entry align="center" valign="middle">8</entry><entry align="center" valign="middle">9</entry><entry align="center" valign="middle">7</entry><entry align="center" valign="middle">10</entry><entry align="center" valign="middle">8</entry><entry align="center" valign="middle">10</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">11</entry></row><row><entry align="center">% Inter-plate CV</entry><entry align="center" valign="middle">11</entry><entry align="center" valign="middle">11</entry><entry align="center" valign="middle">18</entry><entry align="center" valign="middle">23</entry><entry align="center" valign="middle">14</entry><entry align="center" valign="middle">15</entry><entry align="center" valign="middle">15</entry><entry align="center" valign="middle">13</entry><entry align="center" valign="middle">14</entry><entry align="center" valign="middle">16</entry></row></tbody></tgroup></table></tables>

Advantages of the multiplex bDNA assays

0190The above experiments demonstrate that the multiplex bDNA assay is a simple and robust method for gene expression quantification that generates sensitive and reproducible results directly from crude cell lysates, without need for RNA purification, reverse transcription, or target amplifications. One distinctive feature of the assay is its remarkable specificity and accuracy. Cooperative hybridization was demonstrated in a single-plex bDNA assay. Cooperative hybridization can enable exceptionally high assay specificity, enabling multiplexing of the assay. A target mRNA transcript can only be stably captured to its corresponding beads when the transcript binds to multiple CEs. Even when an unintended mRNA transcript binds to one of the CEs in a probe set, the mRNA transcript cannot be stably captured to the beads. Thus, cooperative hybridization can potentially provide two major advantages over simple hybridization currently used in most hybridization based assays: 1) it dramatically reduces the assay background caused by nonspecific cross hybridization; and 2) it enables better discrimination of homologous genes. By leveraging cooperative hybridization in the design of the capture probes, less than 0.1% cross-reactivity has been achieved in all the multiplex panels developed so far, including a rat toxicity panel where several highly homologous cytochrome P450 genes are present. Specificity of the assay is further demonstrated by its excellent spike recovery in the presence of a complex background of total RNA. Because of its exceptional assay specificity, the results of mRNA quantification are therefore highly accurate and reliable.

0191Another distinctive feature of the assay is its simplicity. The assay can quantify RNA transcripts in crude cell lysates and tissue homogenates, as demonstrated in this study using cell lysates from two different cell lines. This feature distinguishes the assay from many other existing mRNA quantification technologies, and makes the assay one of the few that directly measures the mRNA transcripts from their native environment, thus free of any bias imposed by purification procedures and enzymatic reactions. By eliminating the RNA purification, reverse transcription, labeling and amplification steps, assay variations are minimized, leading to significantly improved precision for the overall assay. In addition, since the need for sample preparation is eliminated, the multiplex bDNA assay is well suited for high throughput expression analysis of large sample populations.

0192The third distinctive feature of the assay is its high precision and reproducibility. The intra-plate CV is routinely below 10% and the inter-plate CV routinely below 15% in all experiments where triplicate samples are run, whether the samples are total RNA or cell lysates. The level of consistency in multiplex bDNA assay data is a particularly attractive feature required for quantitative measurement of gene expression in siRNA knockdown, structure-activity relationship, drug dose response, and drug screening applications, where a change of gene expression as small as 10 to 30% needs to be reliably determined and differentiated.

0193Finally, the multiplex bDNA assay is capable of measuring multiple mRNA transcripts simultaneously with good sensitivity and broad dynamic range. The current assay can be expected to detect mRNA transcripts at a concentration as low as 1 copy per cell from a few as 25,000 cells (i.e., the detection sensitivity is 25,000 transcripts), and two mRNA transcripts differing in concentration by as much as 1,000-fold can both be quantified in the same assay with high accuracy. Sensitivity can optionally be increased by decreasing the assay volume (e.g., to 50 µl), including more LEs per probe set so that more amplification multimers bind to each mRNA, and/or using amplification multimers with a higher theoretical fold of amplification. Less than 0.1% cross-reactivity is routinely achieved in more than a dozen multiplex panels tested so far, including cytochrome P450 and ABC transporter panels where several genes with up to 96% homology are present. 30- and 40-plex bDNA assays have been performed, with the same performance characteristics.

0194Although the multiplex bDNA assays described herein simultaneously quantify ten mRNA targets in a single assay, the Luminex technology platform was designed for simultaneous measurement of up to 100 bead analytes. The number of targets in the multiplex bDNA assay can thus be readily increased, e.g., by taking advantage of this platform. Expansion of the multiplex bDNA assay will permit more detailed analysis of mRNA levels in particular pathways and enable more robust systems biology studies. Increased multiplex capability may be desirable for some applications such as microarray validation and follow up studies.

0195The multiplex bDNA assay enables collection of a large amount of data that previously were experimentally challenging to obtain. For example, a previously published study on the cytokine response to inflammatory stimuli was mostly focused on two to three cytokines at a time (<nplcit id="ncit0060" npl-type="s"><text>Hass et al. (1991) "Regulation of TNF-alpha, IL-1 and IL-6 synthesis in differentiating human monoblastoid leukemic U937 cells" Leuk Res. 15:327-39</text></nplcit>, and <nplcit id="ncit0061" npl-type="s"><text>Roberts et al. (1997) "Effects of Porphyromonas gingivalis and Escherichia coli lipopolysaccharides on mononuclear phagocytes" Infect Immun. 65:3248-54</text></nplcit>). With the multiplex bDNA assay, the quantitative expression of dozens of cytokine genes can now be investigated. The multiplex expression analysis of time dependent regulation of apoptotic gene expression upon TNF-alpha treatment in HeLa cells illustrates the potential for high throughput quantitative gene expression analysis. The throughput of the multiplex bDNA assay enables the detailed expression analysis in many samples that received different drug treatments at various dose for different length of times.

0196In summary, the simplicity, sensitivity, accuracy and high reproducibility of the assay, together with the multiplex capability, makes the multiplex bDNA assay a valuable tool, e.g., in applications where accurate and robust quantification of multiple mRNA targets is required, where samples and reagents are precious and limited, and where easy and high throughput sample processing is desired, such as in biomarker validation, compound screening, structure-activity relationship study, toxicity studies, and potentially clinical diagnostics.

Methods

Cooperative hybridization

0197To determine the effect of cooperative hybridization, three oligonucleotide capture probes containing 14, 15, or 16 bases complementary to the CE tails were covalently conjugated to capture plates, respectively, following a published procedure (<nplcit id="ncit0062" npl-type="s"><text>Running and Urdea (1990) "A procedure for productive coupling of synthetic oligonucleotides to polystyrene microtiter wells for hybridization capture" Biotechniques. 8:276-279</text></nplcit>). The sequences of the three capture probes are as follows: 16 base capture probe, 5'-amine-spacer-ACTTTCTTTCCAAGAG-3' (SEQ ID NO:1); 15 base capture probe, 5'-amine-spacer-ACTTTCTTTCCAAGA-3' (SEQ ID NO:2); and 14 base capture probe, 5'-amine-spacer-ACTTTCTTTCCAAG-3' (SEQ ID NO:3). Standard bDNA assays were run on the assay plates coupled with the three capture probes, respectively, using IVT RNA of IL-6 as standard and the bDNA assay probe set for IL-6. To determine the extent of simple hybridization, an oligonucleotide probe (CE-LE) that combines the sequences of the CE tail and the LE tail together was used in bDNA assay in place of the probe set and target RNA. The sequence of the CE-LE probe is as follow: 5'-AGGCATAGGACCCGTGTCtttttCTCTTGGAAAGAAAGT-3' (SEQ ID NO:4), where the series of five small letter t's denotes the linker sequence between the LE tail and the CE tail.

Single-plex bDNA assay

0198Single-plex bDNA assay was performed according to the Instruction Manual of the QuantiGene^® Reagent System (Panomics). Briefly, cells were incubated with Lysis Mixture for 15 min at 37°C in 100µL volume. Probe set for a target gene was added to the cell lysate, transferred to a capture well, and then incubated for 16 hr at 53°C. Unbound material was washed from wells, followed by sequential hybridization at 46°C with branched DNA (bDNA) amplifier and alkaline phosphatase-conjugated label probe. After a final wash, the alkaline phosphatase substrate dioxetane was added to wells and incubated at 46°C for 30 min to develop luminescent signal, which was detected using an Lmax microtiter plate luminometer (Molecular Devices, Sunnyvale, CA).

Capture probes and their coupling to fluorescent-encoded beads

0199Unique sequences of 15 bases were chosen as capture probes. The capture probes were designed to have minimal potential for secondary structure formation or cross-hybridization. They were also screened against homology with sequences of human, mouse or rat genes (BLAST and NCBI databases) or sequences in the bDNA and label probe. Oligonucleotide capture probes were synthesized with 5'-amino linker (BioSearch) and covalently linked to carboxylated fluorescent-encoded microsphere beads (Luminex) following the recommended conjugation procedure from Luminex. Hybridization specificity was evaluated by hybridizing ten coupled microsphere beads to a biotinylated complementary oligonucleotide under the same hybridization conditions as the bDNA assay.

<i>In vitro</i> transcribed RNA (IVT RNA)

0200Complementary DNA clones encoding the full-length target genes were obtained commercially and used as templates for <i>in vitro</i> transcription to generate IVT RNA standards (Invitrogen; Origene; or Open Biosystems). IVT RNA was transcribed for each gene from the restriction enzyme digested plasmid via T3, T7, or SP6 promoters using the Ampliscribe kit (Amersham), quantified using RiboGreen fluorescence (Molecular Probes) and the Envision 2100 Multilabel reader (Perkin Elmer). IVT RNA transcripts for each panel were mixed and serially diluted four-fold to generate standard curve with target RNA levels ranging from 9.6 x 10⁷ to 2.4 x 10⁴ transcripts. The IVT RNA was used as a reference standard to assess the assay sensitivity, specificity, accuracy, and linear dynamic range.

Probe design for single-plex and multiplex bDNA assays

0201Probe design software (<nplcit id="ncit0063" npl-type="s"><text>Bushnell et al. (1999) "ProbeDesigner: for the design of probe sets for branched DNA (bDNA) signal amplification assays Bioinformatics 15:348</text></nplcit>-sus) was modified to design probe sets for target genes in both single-plex and multiplex bDNA assays. For each target sequence, the software algorithm identifies regions that can serve as annealing templates for CEs (5-7 per gene), LEs (10-15 per gene), or BPs. Potential CEs and LEs were examined for possible interactions with other components of the multiplex assay, and CEs and LEs expected to cross-hybridize were not selected for use: CE-LE, CE-bDNA, CE-label probe, and LE-capture probe interactions having highly negative ΔG (e.g., &lt; -7.0 kcal/mole) were removed to minimize non-specific hybridization. Probe sets are essentially the same for both single-plex and multiplex bDNA assay except for the portion of the CE probes that hybridize with capture probe. Several 10-plex panels were developed for the experimental validation. Gene names and reference sequence accession numbers are shown in <b>Table 1;</b> probe sets are shown in <b>Table 3.</b>

Multiplex bDNA assay

0202Multiplex bDNA assay was performed basically as described in the Instruction Manual of the QuantiGene^® Plex Reagent System (Panomics). Briefly, samples containing IVT RNA, total RNA or cell lysates were mixed with the multiplex panel probe sets and the capture beads (about 2000 beads per subset) and hybridized for 16 hours at 53°C in 100µL volume. The components in a 100-µL IVT RNA assay were 33% lysis mixture, 40% capture buffer (Panomics), 1 µg tRNA, and the panel-specific probe set (CE, 0.165 fmol/µl/gene; LE, 0.66 fmol/µl/gene; BP, 0.33fmol/µl/gene). (Hybridization can be performed under any of a variety of suitable conditions; for example, in a solution including the capture diluent described in <nplcit id="ncit0064" npl-type="s"><text>Collins ML et al. (1997) Nucleic Acid Research 25:2979-2984</text></nplcit>: 127 mM LiCl, 5% lithium lauroyl sulfate, 9 mM EDTA, 50 mM HEPES (pH 7.5), 0.05% hespan (DuPont Pharmaceuticals), 0.05% ProClin 300 (Supelco), and 0.2% casein (Research Organics, Hammarsten quality).) Hybridization reactions were transferred to a 0.45-µm filter plate (Millipore, Billerica, MA, USA), followed by sequential hybridization at 46 °C with bDNA amplifier and 5'-dT(biotin)-conjugated label probe. Unbound materials were washed from beads (complexed with probe set and mRNA) through alternating filtration and the addition of wash buffer (0.1xSSC, 0.03% lithium lauryl sulfate). Two washes were performed after each hybridization step. After a final wash, Streptavidin conjugated R-Phycoerythrin (SAPE) was added and was incubated at room temperature for 30 min. The beads were washed to remove unbound SAPE, followed by analysis with Luminex¹⁰⁰IS system (Luminex) or a Bio-Plex system (Bio-Rad). The level of SAPE fluorescence measured from each bead is proportional to the number of mRNA transcripts captured by the bead.

Cell lysates for evaluation of the cytokine panel

0203U937 cells (American Type Culture Collection) at a density around 1-2x10⁵ cells/ml were allowed to differentiate in SFM media (GIBCO) with 100 nM Phorbol-12-myrstyl-13-acetate (PMA, Sigma) for 48 hrs. Differentiated cells, which become adherent, were stimulated with lipopolysaccharide (LPS, Sigma) at a concentration of 1µg/ml in RPMI growth media (GIBCO) with 10% FBS for 4 hours. After cell counting, the cells were directly lysed in the culture flask by the addition of Lysis Mixture to the culture media and incubated at 37°C for 15 minutes.

Cell lysates for evaluation of the apoptosis panel

0204HeLa cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) at an approximate density of 1-2x10⁶ cells/ml and were treated with recombinant human TNFα (1 ng/ml, R&amp;D Systems) or 1% BSA/PBS vehicle control. Both TNFα-treated and vehicle-treated cells were harvested at 1, 3 and 6 hour post-treatment by the addition of Lysis Mixture directly to the culture flasks in order to generate cell lysates suitable for both single-plex and multiplex assays.

Data analysis &amp; statistics

0205Three replicate assays (n=3) were performed for all described experimental samples unless noted otherwise. For all samples, background signal levels in the absence of target mRNAs were determined and subtracted from signals obtained in the presence of target mRNAs. The correlation between the single-plex and multiplex bDNA assay was assessed through matched pair analysis using JMP (SAS Institute). Statistical significance of biological studies was tested using student's t-test or ANOVA where appropriate (P&lt;0.01).