Methods and reagents for preserving RNA in cell and tissue samples

IS 2 255 293 T3

DESCRIPTION

Methods and reagents to preserve RNA in cell and tissue samples.

The present invention relates to the field of molecular biology and provides a novel method and reagent for preserving and protecting ribonucleic acid (RNA) contained in tissue or cell samples from degradation prior to isolating the RNA.

Obtaining high-quality, intact RNA is the first and often most critical step in conducting many fundamental experiments in molecular biology. Intact RNA is required for quantitative and qualitative analysis of RNA expression by Northern blot hybridization, nuclease protection assays, and RT-PCR.

Many reports have been published describing methods for isolating intact RNA from fresh (or quick frozen) cells or tissues. Most of these techniques use a rapid cell disruption step in which tissue is dispersed in a powerful protein denaturation solution containing a chaotropic agent (eg, lithium or guanidinium salt). This rapid disruption of the cell membrane and inactivation of endogenous ribonuclease is critical to prevent RNA from degrading.

To obtain high-quality RNA, it is necessary to minimize the activity of the RNase released during cell lysis and to avoid the degradation of RNA from other sources. This is typically accomplished using isolation methods that disrupt tissues and simultaneously inactivate or inhibit RNases. For specimens with low endogenous ribonuclease, isolation protocols commonly use buffers containing detergents to solubilize membranes, and RNase inhibitors such as placental ribonuclease inhibitor or vanadyl-ribonucleoside complexes. Isolation of RNA from more difficult samples such as intact tissues or cells high in endogenous ribonuclease requires a more aggressive approach. In these cases, the tissue or cells are rapidly homogenized in a potent protein denaturant (usually guanidinium isothiocyanate), to irreversibly inactivate nucleases and solubilize cell membranes. If a tissue sample cannot be homogenized immediately, it should be quick frozen by immersion in liquid nitrogen, and stored at -80 ° C. Samples frozen in this way should never be thawed before isolating the RNA or the RNase released during cell lysis that occurs during freezing will rapidly degrade RNA. The tissue should be immersed in a liquid nitrogen bath and ground to a fine powder using a mortar and pestle. Once pulverized, the still frozen tissue is homogenized in a buffer for RNA extraction. In the laboratory, rapid freezing of samples in order to delay RNA extraction has the drawback of substantially increasing manual processing time. Processing multiple samples with liquid nitrogen and mortar and pestle is extremely time consuming.

Quick freezing is even less convenient outside of the laboratory setting, but is still considered necessary by those working in the field. Scientists collecting specimens in the field for analysis do not have access to high speed homogenizers. They are forced to carry a supply of liquid nitrogen or dry ice large enough to store samples until they can be transferred to an ultra-low temperature freezer. Similarly, RNA extracted from human biopsy samples is usually partially or mostly degraded because specimens are not routinely quickly frozen by pathologists to preserve RNA.

There have been attempts to isolate RNA from archival samples that had not been prepared by the flash freeze methodology. For example, Esser et al., 1995 claim the isolation of full length RNA from cells fixed with 5% acetic acid, 95% ethanol, with RNase inhibitors. However, in this publication, isolated cells in suspension were fixed with an acetic acid / ethanol solution at -20 ° C and then kept at 4 ° C for a relatively short time. Unfortunately, the study conducted by the Inventor showed that Esser et al .'s 95% ethanol / 5% acetic acid solution does not meet the quality standards required by the present invention. RNA recovered from both tissue samples and spleen cells in suspension kept at 4 ° C for 20 hours was partially degraded, while RNA isolated from tissues stored at room temperature was completely degraded. The experiments reported by Esser et al. show that the method results in a loss of RNA, due to losses caused by ethanol in the cells. Using that method, 70% of the RNA is lost immediately during fixation, and after one hour, 80% of the RNA is gone. Furthermore, in an assay in which spleen cell and tissue samples were stored in the 95% ethanol / 5% acetic acid solution overnight at 25 ° C, the RNA of both the tissue and cell samples was completely degraded. The data is shown in Fig. 1.

The use of intact, high-purity RNA is essential to perform various molecular biology assays and experiments such as Northern blot hybridization, nuclease protection assays, RT-PCR, and medical diagnostics. The intrinsic instability of RNA and the presence of RNases in samples makes isolating intact RNA a difficult procedure. Furthermore, the isolation and titration of samples containing RNA is typically tedious and time consuming. Contamination of a molecular biology laboratory with RNases due to human error can have catastrophic results. Therefore, there is a progressive need to develop improved techniques, to make RNA isolation and assay methods more sensitive, more specific, faster, easier to use, and less susceptible to human error and manipulation. Therefore, in many cases it would be advantageous to use an automated RNA preservation protocol in research facilities.

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For example, the present invention could be combined with rapid RNA assay techniques or integrated nucleic acid diagnostic devices (US Patent 5,726,012, US Patent 5,922,591, incorporated by reference) to an automated and efficient RNA analysis and preservation.

US Patent 5,256,571 reports a cell preservation solution comprising a water miscible alcohol in an amount sufficient to bind mammalian cells, an anti-caking agent, and a buffering agent. At least one publication, Dimulescu et al., Reports the apparent use of this fixative to preserve cervical cancer cells and cord blood lymphocytes prior to isolating RNA.

A large body of literature suggests that ethanol and acetone combinations are the best known fixatives for future recoveries of nucleic acids from archived tissues. However, in view of the inventors' studies, such an ethanol / acetone mixture does not provide all the desired characteristics in an RNA preservation medium. The mixtures do not protect RNA at room temperature, do not allow RNA to be preserved in solid, multi-cell samples, and are also flammable, making them inherently less attractive as a general purpose reagent.

There is some peripherally related technique that addresses the aspects of preservation or recovery of RNA from samples of preserved or fixed tissue. These reports include numerous evaluations of the suitability of histological fixatives to maximize the signal obtained by in situ hybridization to detect (not recover) RNA in tissue samples (eg, US Patent Nos. 5,196,182 and 5,260. 048). Other reports detail methods for recovering fragmented RNA from fixed tissues for limited molecular analysis by PCR.^TM(Koopman et al., Foss et al., Stanta et al., Houze et al.). To recover this fragmented RNA, samples are typically treated with proteinase K to degrade the structural components of the tissue, then the RNA is extracted with a guanidinium-based solution. RNA recovered from fixed tissue is of extremely poor quality, with an average size of approximately 200 bases (Stanta 1991). This is probably due to several factors including the action of endogenous RNase and the cross-linking of RNA into the intracellular matrix during fixation. Since RNA is largely degraded, it cannot be used in Northern analysis or nuclease protection assays. It can be used in RT-PCR, but only for the amplification of very small fragments.

The use of ammonium sulfate to precipitate proteins in solution is known, but the use of ammonium sulfate to preserve RNA is not known in the art to the best of the inventor's knowledge. Two reports describe the use of ammonium sulfate to investigate the folding and activity of mammalian ribonuclease A (Allewell et al. And Lin et al.). Allewell et al. investigated the effects of ammonium sulfate on the folding and activity of RNase A. At pH 5.5, ribonuclease A activity is suppressed to approximately 10% of the level of untreated controls over a wide range of ammonium sulfate concentrations. This suppression of activity was expected by the authors. It appears to be due to salt-induced protein denaturation. Unfortunately, even 10% RNase activity would substantially degrade RNA in a sample over time. Therefore, this inhibition is not sufficient to protect RNA in many applications. When ammonium sulfate is at pH 7.0, RNA loop A activity is suppressed at low concentrations as expected, but unexpectedly increases to 110% of the level of the untreated control at higher concentrations (3M). The authors theorize that the combination of neutral pH and high salt concentrations forces a refolding of the protein into an alternative, highly active configuration. However, the group of Allewell et al. I was examining pure RNase activity in solution, not in a cell sample containing many RNases.

In view of the above, there is a need for methods and reagents that allow the preservation and recovery of high quality, intact RNA from tissue samples stored at or near room temperature.

The present invention relates to a novel method and reagents for preserving RNA in tissue fragments at temperatures above the freezing point of preservatives for long periods of time, including days to months, prior to isolating the RNA. There are no previous reports that disclose a reagent or method similar to that described in this application. This advance decreases the need to either immediately process samples to extract RNA, or the restriction of only isolating tissue at sites that have a supply of liquid nitrogen or dry ice.

The present invention relates to methods for conserving RNA comprising: (1) obtaining a sample containing RNA; and (2) treating the sample with an RNA preservation medium that infiltrates the sample, and protects the RNA from nucleases. In a preferred embodiment, the RNA preservation medium causes precipitation of the RNA in the sample along with the cellular proteins in the sample. This coprecipitation of RNA and cellular proteins is believed to generate RNA physically inaccessible to nucleases, while the action of the RNA preservation medium simultaneously inactivates or inhibits the action of nucleases.

The RNA preservation medium comprises a salt, at a concentration from 40 g / 100 ml to the saturation concentration of the salt, which precipitates the RNA in the sample together with the cellular proteins. The salt is ammonium sulfate or cesium sulfate. In currently preferred commercial embodiments, the salt is ammonium sulfate.

Specifically, salt concentrations of 40, 55, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, can be used.

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110, 120, 130, 140 or 150 mg / ml, and the concentration can be in a defined range between any pair of these concentrations.

Of course, during use, some dilution of the salt concentration may occur due to, for example, the sample containing liquids. Therefore, these salt concentrations may be higher than the final salt concentrations obtained in some uses. Furthermore, it is contemplated with respect to the present invention that salt concentrations in excess of the saturation concentration may be used. In such embodiments, there may be salt that is not dissolved in the RNA storage medium. This should not affect the RNA storage capacity of the media. In fact, media that have more than one saturation concentration of a salt may be of some use in applications where the media is added to a liquid sample. In such cases, during the addition to the liquid sample, the salt that is not dissolved before the addition, may dissolve due to the increase in volume of the liquid. Therefore, the final concentration of a salt may be at a higher level than would be possible if a preservation medium were used that contained a salt concentration at or below saturation level.

In a preferred commercial embodiment, the salt is ammonium sulfate in a concentration of 70 g / 100 ml.

The present invention is not limited to the use of ammonium sulfate, and cesium sulfate will also be useful for RNA protection in tissue samples and cell samples, due to the following reasons. The solubility of individual proteins is highly dependent on the pH and salt concentration of the aqueous medium. Virtually all proteins are insoluble in pure water. As the ionic strength of the medium increases, the proteins become more soluble. This is known as "salting in" of proteins. Above a certain ionic strength, the solubility of the protein decreases. The precise conditions under which this happens are unique to each protein / salt combination. In fact, at some salt concentrations, one protein can be completely insoluble while another is at its maximum solubility. This phenomenon is known as “salting out”. The present means of RNA protection are believed to work due to the "salting out" effect of high levels of salt. The theory is that the "salting out" of proteins present in cells from tissue samples or cell samples is what leads to the formation of RNA-protective RNA / protein complexes. The importance of the phenomenon "salting out" in this application increases several times. First, this highlights that the efficiency of RNA protection by protein precipitation using high salt concentrations is complex and that certain combinations of ionic strength (salt concentrations) and pH can make a particular salt much more effective in a formulation than at a different pH or concentration. Second, it provides a sound scientific foundation for the basic mechanism of action of the reagents in this application, and provides guidance in the search for additional RNA protection compounds within the scope of the invention. In order to determine whether another salt or putative RNA protective compound will work in the methods and reagents of the invention, it is simply necessary to obtain that salt or compound and test it as described in the examples. By following the teachings of the examples, a person of knowledge can easily elucidate whether a candidate substance is really an RNA protective compound. Third, based on the theory that the precipitation of intracellular proteins is the key to protecting RNA in situ, this explains why alcohol and acetone (agents that can also precipitate proteins, albeit by a different mechanism), are partially active in protecting RNA in tissues, although they do not protect as much as is necessary for most applications.

In some embodiments, the RNA preservation media will comprise a combination of two salts that precipitate the RNA in the mixture along with the cellular protein.

The RNA preservation medium may further comprise ethanol, methanol, acetone, trichloroacetic acid, 1-propanol, 2-propanol, polyethylene glycol or acetic acid. These additional potential protective components can precipitate proteins in conserved cells and thus protect RNA. However, these potential additional components are not salts. It is anticipated that in some embodiments, these organic solvents will be used in combination with a concentration of salt to obtain one of the RNA preservation media as described herein.

In some embodiments, the RNA preservation medium comprises a salt such as ammonium sulfate, cesium sulfate, methanol, trichloroacetic acid, 1-propanol, 2-propanol, polyethylene glycol, or acetic acid. Furthermore, the RNA preservation medium may comprise a chelator of divalent cations, for example EDTA.

Typically, the RNA storage medium comprises a buffer so that a constant pH can be maintained. For example, the buffer can be sodium citrate, sodium acetate, potassium citrate, or potassium acetate. In a currently preferred commercial embodiment, the buffer is sodium acetate. Typically, the RNA preservation medium has a pH between 4 and 8. In currently preferred commercial embodiments, the pH is 5.2.

The sample preserved in RNA preservation media can be any one of several types of samples. For example, the sample can be a suspension of cells, such as bone marrow aspirates, leukocytes, sperm, blood, serum, plasma, bacteria, cells from tissue cultures, or algae. Alternatively, the sample can be solid tissue, eg, the tissue sample is from brain, heart, liver, spleen, thymus, kidney, testis, ovary, tumors, tissue from biopsies, plant shoots, roots, or leaves. In some cases, the sample may comprise a complete organism. For example, the organism can be a fish, insect, tadpole, coral, or embryo. In some protocols, it will be beneficial to keep an organism or mixture in the RNA preservation medium during dissection. For example, it might be beneficial to dissect an organism in the RNA preservation medium when the sample

ES 2 255 293 T3 comprises an organism that is a pathogen within a mixture of tissue or other organism. In this way, the RNA of the pathogen can be conserved. In addition, RNA from the tissue sample or other organism is preserved.

In many preferred methods, the practice of the invention further comprises the step of isolating the conserved RNA. One of the advantages of the present means of RNA preservation is that RNA can be isolated from tissue at a higher temperature than allowed in prior techniques. For example, RNA can be isolated at a temperature that is greater than -20 ° C. In fact, RNA can be isolated at room temperature.

In some cases, the sample can be stored in the RNA preservation medium prior to isolating the RNA. For example, tissue is stored unfrozen at -20 ° C to 45 ° C. Due to the salt content of some RNA storage media, mixtures do not freeze at -20 ° C. In preferred embodiments the sample can be stored above 0 ° C.

In other embodiments of the present invention, a method of preserving RNA will comprise obtaining an RNA-containing mixture, providing a salt, and mixing the sample and the salt in a liquid to form an RNA preservation composition that infiltrates the sample and protects the Nuclease RNA. In one embodiment, the sample is comprised in the liquid prior to mixing the sample with the salt. In another embodiment, the salt is in a solid form prior to mixing the sample and the liquid. In yet another embodiment, the salt is comprised in the liquid prior to mixing the sample with the salt.

In one embodiment of the invention, the sample is blood cells and the liquid is blood serum. In another embodiment the sample is urine. In other embodiments, the liquid is water. In still other embodiments, the liquid is a buffer.

In some embodiments, the RNA preservation compositions comprise obtaining a sample containing RNA, providing a salt, and mixing the sample and the salt in a liquid. The liquid can be a component of the sample, or it can be added to the sample and salt. The salt is typically present in a concentration sufficient to precipitate the RNA in the sample along with the cellular protein. In some cases, adding a highly concentrated salt or a saturated salt to a liquid sample will suffice. Adding salt that can result in a salt concentration greater than the saturation concentration to the final liquid sample has the advantages of allowing conservation concentrations of RNA to be reached quickly and avoiding the need to carefully consider the amount of salt required to reach a specific concentration in a given sample. Furthermore, any salt that does not dissolve will not affect the RNA preservation properties of the composition.

In another embodiment, the RNA preservation composition comprises a divalent cation chelator. In still other embodiments, the RNA preservation composition comprises a buffer, wherein said buffer has a pH between 4 and 8.

The solid components of the present invention (ie, salts, buffers, and protectants) can be prepared to generate the desired final concentrations of component in solution when added to an aqueous sample. The solid components can further be provided as powders, tablets, pills, or other suitable formulations that provide the desired properties of an RNA preservation composition. Solid components can be added directly to a sample, added to a sample / liquid mixture, or be present in a collection container prior to collecting a sample or sample / liquid mixture. In the formulation of powders, tablets and pills, the addition of excipients and bulking agents such as mannitol, lactose, starch, cellulose, and the like, is also considered to provide the desired solid characteristics (i.e. better solubility, storage stability , particle dispersion). The solid components of the present invention can be added prior to sample collection, after sample collection, or any combination of these.

In one embodiment of the invention, pre-measured aliquots of a solid or liquid RNA preservation composition can be loaded into sample collection containers and an appropriate volume of an RNA-containing sample can be added. The collection container will then be shaken, dissolving any solid components of the RNA preservation composition, minimizing operator exposure to an RNA sample. For example, a solid RNA preservation composition could be any of the salts previously described. Thus, in particular embodiments of the invention, stabilization of RNA in a biological specimen such as blood and urine is contemplated as described above.

In one example, a vial can be supplied to collect a specimen such as urine or blood with previously measured aliquots of an RNA preservation composition. Immediately after collecting said specimen, the vial could be shaken, mixing the specimen containing the RNA (ie, sample) with the RNA preservation composition. An empty blood collection vial comprising an outwardly projecting needle is contemplated by us in the present invention (US Patent 5,090,420, specifically incorporated herein by reference in its entirety) for rapid collection. and sample mix containing RNA. In one embodiment, automated RNA conservation is contemplated. Automated RNA conservation methods will be less tedious, fast, easy to use, and less susceptible to human error and manipulation.

Clinical specimen collection kits adapted to be sent by mail are also contemplated (Patent

US 2 255 293 T3 5,921,396, specifically incorporated herein by reference in its entirety) for use with previously measured aliquots of RNA preservation compositions of the present invention. Samples containing RNA may be collected (for example, off-site) and mixed with previously measured aliquots of an RNA preservation composition provided in vials, preserving the RNA for shipment to a suitable RNA testing site.

Alternatively, an RNA preservation composition can be compressed into a tablet or pill and stored in bulk. The tablets will be a convenient composition to store and can be added in the correct quantity to a sample of any size in any type of container. Therefore, in other types of embodiments of the present invention, RNA preservation components in the form of tablets or pills are contemplated for collection of samples in the field from sources such as water reservoirs, sewage plants or the dairy industry. In some instances, the RNA preservation media comprises predetermined final salt concentrations or supersaturated salts that can be supplied as packages. Containers of supersaturated salt or RNA preservation salt will be especially useful in field studies, as there may be limited space or resources for analytical equipment. For example, containers can be supplied as pre-measured and packaged aliquots for a 1mL, 5 mL, 10 mL sample. Of course, any size of container can be provided to contain various amounts of salt as an anhydrous powder, a hydrated powder, or as a powder and liquid, individually packaged or a combination thereof. Therefore, to preserve RNA in a sample, one would simply have to add the contents of the container to a sample and mix.

Some advantages of using a solid component RNA preservation composition would be the weight savings in storage and transportation, that solid component spills are less likely, and savings from the lower volume (i.e., keeping samples with dry reagents will minimize the final sample volume).

In other embodiments of the invention, the RNA preservation compositions further comprise isolating the conserved RNA, wherein the RNA is isolated at a temperature that is greater than -20 ° C. In other embodiments, the sample is stored prior to isolating the RNA, in which the sample is stored at temperatures greater than 0 ° C.

The inventor's research indicates that 3M ammonium sulfate at pH 7.0 is fully effective in preserving intact RNA in intact tissue samples at all but extreme temperatures (37 ° C-42 ° C). It is proposed that during application to the sample, ammonium sulfate diffuses within tissue and cells and causes precipitation of cellular proteins (probably along with RNA that is closely associated with many different proteins in vivo) into protected complexes. . Furthermore, RNase, which is localized in cytoplasmic vesicles, can also precipitate and become inaccessible to cellular RNA.

It is believed that the mode of action of the claimed invention and the mode of action of the mixture of Allewell et al. They are different. If the mode of action of the inventive solutions were the same as that described by Allewell et al. one would expect RNA isolated from tissues stored in the buffers of the invention to be degraded. Allewell et al. report that at pH 5 RNase is more active than normal. Therefore, if RNAlater ™ acted by the same mechanism, one would expect an increase in RNase activity. Obviously, this will limit the ability of RNA preservation media to conserve RNA. Based on the observation that such degradation does not occur, the present invention operates by a different mechanism than that described by Allewell et al.

The term "RNAlater ™" is a registered trademark of Ambion, Inc., for certain commercial formulations of RNA preservation media as disclosed herein. In general, the term "RNAlater ™" is used to denote the formulation disclosed in Example 2, which is composed of 25 mM sodium citrate, 10 mM EDTA, 70 g of ammonium sulfate / 100 ml of solution, pH 5, 2. This reagent works by rapidly infiltrating cells with a high concentration of ammonium sulfate, producing a massive precipitation of cellular proteins, the cellular structure remains intact. The advantage of this is that the cells can be preserved and still be identified histologically.

Following long-established patent practice, the words "a" and "an" when used in conjunction with the word "comprise" in the claims or specification, denote one or more.

The following drawings are part of the present specification and are included to better demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of those drawings in combination with the detailed description of the embodiments as presented herein.

Fig. 1

Alcohol and acetone are inconvenient for preserving cell samples

RNA isolated from isolated mouse liver stored overnight at 4 ° C (lanes 1-5) or 37 ° C (lanes 610). Lanes 1 and 6, storage in ethanol. Lanes 2 and 7, storage in acidified ethanol pH 4.0. Lanes 3 and 8, storage in acetone. Lanes 4 and 9, storage in acidified acetone pH 4.0. Lanes 5 and 10, RNAlater ™ storage.

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Fig. 2

RNA is labile in fresh tissue samples

Fresh samples of mouse liver (lanes 1 and 2), testis (lanes 3 and 4), and spleen (lanes 5 and 6) were placed in RNAlater ™ (lanes 2, 4, and 6) (25 mM sodium citrate, 10 mM EDTA, 70 gm Ammonium Sulfate / 100 ml solution, pH 5.0), in an RNA extraction solution based on 4 molar guanidinium isothiocyanate (GITC) (lanes 1, 3, and 5) at 4 ° C for 12 hours. RNA was extracted and analyzed by gel electrophoresis. Intact RNA was only observed in lanes corresponding to tissues conserved in RNAlater ™ (lanes 2, 4 and 6).

Fig. 3

Defining the effective concentration range of ammonium sulfate that protects RNA in tissue

Freshly obtained mouse liver fragments were placed on RNAlater ™ with various amounts of ammonium sulfate. The samples contained 0, 10%, 20%, 30%, 40%, 50% or 70% ammonium sulfate (lanes 17 respectively). After incubation at 4 ° C for 24 hours, RNA was extracted from the sample and analyzed by denaturing electrophoresis on agarose gels.

Fig. 4

Improved potency of RNAlater ™ at extreme temperatures by optimizing pH and ammonium sulfate concentrations

The effects of pH and ammonium sulfate were evaluated at extreme temperatures. Fresh mouse liver was stored at room temperature or 37 ° C for 24 hours in 4 formulations of the RNAlater ™ solution. Lane 1: pH 7.0, ammonium sulfate 70 g / 100 ml, Lane 2: pH 5.0, ammonium sulfate 55 g / 100 ml, Lane 3: the original formulation of RNAlater ™ (containing ammonium sulfate 55 g / 100 ml at pH 7.0), Lane 4: pH 5.0, ammonium sulfate concentration 70 g / 100 ml.

Fig. 5

The specificity of ammonium sulfate in the efficacy of RNAlater ™

Figure 5A and 5B: Fresh liver samples were incubated in 5 buffers for 3 days at 25 ° C (Fig. 5A) and 37 ° C (Fig. 5B), lane 1; RNAlater ™ with ammonium carbonate, lane 2; with ammonium chloride, lane 3; with potassium sulfate, lane 4; with magnesium sulfate, lane 5; ammonium sulphate. Fig. 5C: Fresh liver incubated overnight at 4 ° C in RNAlater ™ containing: lane 1, cesium chloride; Lane 2, cesium sulfate; lane 3, ammonium sulfate.

Fig. 6

RNA isolated from mammalian tissues stored at 4 ° C in RNAlater ™

Fresh mouse brain, heart, kidney, liver and spleen (lanes 1-5) were placed in the RNAlater ™ solution and stored at 4 ° C. After incubation for one week (Fig. 6A), 2 weeks (Fig. 6B), and 4 weeks (Fig. 6C), RNA was extracted from tissue samples and analyzed by denaturing electrophoresis on agarose gels.

Fig. 7

RNA isolated from mammalian tissues stored at room temperature (25 ° C) in RNAlater ™

Fresh mouse brain, heart, kidney, liver and spleen (lanes 1-5) were placed in the RNAlater ™ solution and stored at 25 ° C (room temperature). After incubation for two weeks (Fig. 7B) or four weeks (Fig. 7C), RNA was extracted from tissue samples and analyzed by denaturing electrophoresis on agarose gels.

Fig. 8

RNA isolated from mammalian tissues stored at extreme temperatures (37 ° C) in RNAlater ™

Fresh mouse liver, kidney and spleen were placed in the RNAlater ™ solution and stored at 37 ° C. After incubation for three days, RNA was extracted from tissue samples and analyzed by denaturing electrophoresis on agarose gels.

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Fig. 9

Using Ambion to obtain RNA from tissues preserved in RNAlater ™

In lanes 1-3 there is liver, heart and kidney, ToTally RNA ™, in lanes 4-6, RNAqueous ™. Fresh samples of mouse liver (lanes 1 and 4), heart (lanes 2 and 5), and kidney (lanes 3 and 6) were placed in RNAlater ™ and stored overnight at 4 ° C. RNA was isolated from samples of the same size using either the ToTally RNA ™ kit from Ambion (Lanes 1-3) or the RNAqueous ™ kit from Ambion (Lanes 4-6). Lane 1 - molecular weight marker.

The present invention relates to the field of molecular biology and provides new methods and reagents for preserving and protecting ribonucleic acid (RNA) contained in RNA-containing samples prior to RNA isolation from degradation. Surprisingly, this is accomplished without storing at ultra-low temperatures or breaking the samples. For example, tissue isolated from a human biopsy can be placed in RNA preservation medium and stored refrigerated for a long period of time, until the investigator has time to extract the RNA for analysis.

The following examples illustrate the utility of the present invention. All examples use fresh animal or plant tissues, living cells in suspension, or other samples containing RNA. The methods and compositions are applied to RNA conservation in a wide variety of bacterial, plant, and animal species, including humans. RNA samples recovered in these experiments are analyzed by electrophoresis on formaldehyde / agarose gels, stained with ethidium bromide, and illumination with 300 nm ultraviolet light, as described in Molecular Cloning, A Laboratory Manual (Maniatis, et al) .

Examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art should appreciate that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to work well within the practice of the invention, and therefore may be considered to constitute preferred modes of practice.

Example 1

Criteria for analyzing RNA to determine if it is "intact"

The Inventor routinely performs RNA analyzes designed to assess whether such samples are intact. This criterion was used in the examples that follow to objectively determine the quality of RNA recovered from tissues preserved in RNAlater ™, other inventive RNA preservation media, or other solutions.

RNA is analyzed by electrophoresis on agarose formaldehyde gels using the basic protocol described in "Molecular Cloning, a Laboratory Manual" (Maniatis, Fritsch, Sambrook, eds. Cold Spring Harbor Press). In order to visualize the RNA, ethidium bromide was added to the gel, for staining by intercalation. Ethidium bromide, when embedded in nucleic acid, emits fluorescent light under ultraviolet light, allowing nucleic acid to be visualized. Intact RNA appears as a heterogeneous mRNA smear (from 0.5 kilobases to 10 kilobases), with two very prominent, discrete bands (28S and 18S ribosomal RNA), superimposed on a background smear. in a ratio of 2: 1. There should be very little evidence of discrete bands of intermediate size between 18S and 28S RNA. Partially degraded RNA is characterized by a loss of heterogeneous high molecular weight RNA, multiple smaller breakage products of ribosomal RNA, and a 2: 1 deviation of 28S versus 18S (28S is more sensitive to degradation ). Severely degraded RNA will have 28S: 18S ratios less than 1: 1, and will show a smear of degraded ribosomal RNA from 2 kilobases to <0.1 kilobases.

"Partially degraded" as used in this specification means that the 28S: 18S rRNA ratio is aberrant and can be as low as 1: 1, but the rRNA bands are still distinguishable.

"Mostly degraded" as used in this specification means that the ratio of 28S: 18S is less than 1: 1. The 28S may be barely visible, but there is still nucleic acid in a smear that extends from the approximate position of the 28S rRNA downward.

"Completely degraded" and "degraded" as used in this specification means that the only RNA present is in a low molecular weight smear below the normal position of 18S rRNA.

Example 2

Preparation of an RNAlater ™ RNA Preservation Medium as an Example

The description of this example provides one way in which RNAlater ™ can be prepared. First, the following stock solutions and reagents should be obtained or prepared: 0.5 M disodium EDTA dihydrate (18.61 g / 100 ml, adjust the pH to 8.0 with NaOH while stirring); trisodium citrate salt, 1M dihydrate (29.4 g / 100 ml, with stirring until dissolved); Ammonium sulfate, powder; sterile water.

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In a beaker, combine 40 ml of 0.5 M EDTA, 25 ml of 1 M sodium citrate, 700 g of ammonium sulfate, and 935 ml of sterile distilled water, mix on a hot plate shaker at low heat until that the ammonium sulfate dissolves completely. Let cool, adjust the pH of the solution to pH 5.2 using H₂SW₄ 1 M. Transfer to a screw cap bottle and store at room temperature or refrigerated.

Example 3

Example of a general way of preserving tissues in RNA preservation media

Tissue samples to be preserved in RNAlater ™ or other inventive RNA preservation media should be separated from the source as quickly as possible and placed in RNAlater ™ or other inventive RNA preservation media. Some tissue samples may have a protective membrane or other barrier that will prevent rapid infusion of the RNAlater ™ or other RNA preservation media of the invention, such as a waxy coating on a leaf or the capsule of a kidney or testis. These protective barriers should be broken to allow rapid infiltration of RNAlater ™ or other inventive RNA preservation media into the sample. In addition, large samples should be dissected into smaller fragments to maximize diffusion. As a general guide, the thickness of the specimen will be limited to 0.5 cm in at least two dimensions. Samples consisting of cells in suspension should be concentrated to a small volume by gently centrifuging at sufficient g-force to concentrate cells without damaging them, resuspend them in a minimal volume of removed supernatant, and then mix with 5 volumes of RNAlater ™ u other inventive RNA preservation media (v / v). If concentration is not possible, the cell suspension should be diluted in 10 volumes of RNAlater ™ or other inventive RNA preservation media. Alternatively, any other volume of liquid, essential medium, or amount of a solid RNA preservation composition can be used that results in preservation of the RNA. Since the buffer will not kill the cells, concentration by centrifugation can be done later.

Samples that will be stored for a week or less can be stored at room temperature (25 ° C). For longer stability, samples should be stored refrigerated. For permanent archival storage (months - years), samples can be stored in a standard freezer (-20 ° C). To isolate RNA from treated samples, tissue samples must be transferred directly into a tissue extraction buffer. Cells in suspension should be concentrated by centrifugation, then suspended in a centrifugation buffer and processed.

Example 4

RNA is labile in fresh tissue samples

Fresh mouse liver, testis, and spleen samples (~ 0.5 cm³) in RNAlater ™ or in a 4 M guanidinium isothiocyanate (GITC) based RNA extraction solution, or water at 4 ° C for 12 hours. This guanidinium solution is a typical RNA extraction buffer (and is found in the commercially available Ambion ToTally RNA ™ and RNAqueous ™ kits). GITC is a powerful chaotropic agent used either alone or in conjunction with other agents in virtually all RNA isolation protocols. After an overnight incubation, RNA was extracted from tissue samples and analyzed by denaturing electrophoresis on agarose gels. Intact RNA was only observed in the lanes corresponding to the tissues conserved in RNAlater ™. RNA extracted from samples conserved in GITC was highly degraded. Therefore, GITC does not preserve RNA in intact tissue samples. Animal tissues stored in water or biological buffers such as normal saline do not produce detectable RNA after identical overnight incubation. The data is shown in Fig. 2.

Example 5

Determination of the effective concentration of ammonium sulfate that protects RNA in tissues

Freshly isolated mouse liver was placed on various media that presumably conserves RNA with various concentrations of ammonium sulfate. The samples contained 0, 10%, 20%, 30%, 40%, 50%, or 70% ammonium sulfate. After incubation at 4 ° C for 24 hours, RNA was extracted from the sample and analyzed by denaturing electrophoresis on agarose gels.

In the sample that did not contain ammonium sulfate, all that was observed was a low molecular weight smear. No specific ribosomal RNA band was observed. At an ammonium sulfate concentration of 10%, slight evidence of rRNA bands was observed. At 20% ammonium sulfate, the 18S band is more apparent, and there is a degraded 28S rRNA smear extending below the predicted position for 28S rRNA. No specific band is observed for 28S. At 30% ammonium sulfate, both the 28S and 18S rRNA bands are visible, however there is extensive degradation (based on the aberrant 28S: 18S ratio and the numerous smaller bands observed). The sample with 40% ammonium sulfate appears almost intact and the yield is 10 times higher than with 30%. While these experiments suggest a minimum requirement of 30-40% ammonium sulfate for RNA protection in tissues, the effective concentration will depend on tissue type, tissue fragment size, storage temperature, and shelf life. storage.

IS 2 255 293 T3

Under more severe incubation conditions, a higher concentration of ammonium sulfate is necessary for RNA preservation. For example, for the preservation of RNA in tissue samples at 25 ° C, 55 g / 100 ml are needed, as described in Example 6 for maximum protection in tissue samples at 37 ° C, 70 g / 100 ml seem to be necessary . Furthermore, if samples are stored in 55g or below 37 degree ammonium sulfate for 24 hours, at least some studies produce partially degraded RNA as can be inferred from a 1: 1 ratio of 28S: 18S rRNA. The data is shown in Fig. 3.

Example 6

Improving the potency of RNA storage media at extreme temperatures by optimizing pH and ammonium sulfate concentrations

The effect of pH and ammonium sulfate was evaluated at extreme temperatures. Fresh mouse liver was placed in 4 formulations of the RNA preservation media under study and stored at room temperature, or 37 ° C, for 24 hours. RNA was extracted from tissue samples and analyzed by denaturing electrophoresis on agarose gels. The formulation containing 55 g / 100 ml of ammonium sulfate at pH 7.0 was effective at room temperature, but did not completely protect the RNA at 37 ° C. A combination of low pH (5.0) and higher ammonium sulfate concentration (70 g / 100 ml) was much more effective at high temperatures than the original formulation and provided intact RNA after 3 days at 37 ° C. No modification alone (low pH or higher concentration of ammonium sulfate) appreciably improved RNA stability over and above that provided by the original formulation. The data is shown in Fig. 4.

Example 7

The specificity of ammonium sulfate on the efficacy of RNA preservation media

Four additional formulations of RNA preservation media were produced for study. Ammonium sulfate is replaced by saturating amounts of ammonium carbonate, ammonium chloride, potassium sulfate, or magnesium sulfate. Fresh liver samples were incubated in these buffers for 3 days at 25 ° C and 37 ° C. RNA was extracted and analyzed by denaturing electrophoresis on agarose gels. Only the ammonium sulfate control prevented the RNA from degrading. Maximum protection appears to require high concentrations of both sulfate and ammonium ions. Ammonium sulfate is probably the most effective of the salts studied because it is much more soluble in water than these other salts, allowing formulations with a very high salt content. This hypothesis was challenged by evaluating two (more expensive) salts: cesium sulfate (which is very soluble - saturation at 362 g / 100 ml) and cesium chloride (70 g / 100 ml). These salts were substitutes (in equal masses) for ammonium sulfate in the solution described in Example 2 and the liver samples were processed as indicated above. Cesium sulfate and cesium chloride offered partial protection to liver RNA at 4 ° C. Taken together, the data suggest that of the salts studied, ammonium sulfate has the greatest ability to protect RNA from degradation in intact tissues. However, the partial protection that the inventors see with high concentrations of another salt suggests a shared mechanism of action, with varying degrees of efficiency. The data is shown in Fig. 5.

Example 8

RNA isolated from mammalian tissues stored at 4 ° C in RNAlater ™

Fresh mouse brain, heart, kidney, liver and spleen samples were placed in RNAlater ™ solution, prepared as explained in Example 2, and stored at 4 ° C. After incubation for one week, 2 weeks, and four weeks, RNA was extracted from tissue samples and analyzed by denaturing electrophoresis on agarose gels. All RNA samples were intact. Fresh mouse brain, kidney, liver and spleen samples were placed in 10 volumes of RNAlater ™ and stored at 4 ° C. After incubation for one week, two weeks, and four weeks, equivalent weight fragments of tissue were removed and processed. Each tissue sample was placed in a guanidinium isothiocyanate lytic solution, homogenized and isolated using the RNAqueous ™ kit from Ambion (as described in Example 20). The RNA concentration was determined by measuring the absorbance at OD 26o. RNA was analyzed by electrophoresis on formaldehyde-agarose gels as described in Example 1.

RNA samples were considered "intact" based on the presence of clean RNA ribosomal bands, defined in a 2: 1 ratio of 28S: 18S. Furthermore, overall quality was judged by a heterogeneous RNA background smear and the lack of visible ribosomal RNA cleavage products. The data is shown in Fig. 6. Example 9

RNA isolated from mammalian tissues stored at room temperature (25 ° C) in RNAlater ™

Fresh mouse brain, heart, kidney, liver and spleen samples were placed in the RNAlater ™ solution and stored at 25 ° C (room temperature). After incubation for two to four weeks, RNA was extracted from tissue samples and analyzed by denaturing electrophoresis on agarose gels as described above. The RNA recovered after two weeks was intact. RNA recovered after one month

Incubation ES 2 255 293 T3 was still ~ 50% intact judging from the appearance of the 18S and 28S ribosomal bands. The data is shown in Fig. 7.

Example 10

RNA isolated from mammalian tissues stored at extreme temperatures (37 ° C) in RNAlater ™

Fresh mouse liver, kidney and spleen samples were placed in the RNAlater ™ solution and stored at 37 ° C. After incubation for three days, RNA was extracted from the tissue samples and analyzed by denaturing electrophoresis on agarose gels, as described above. The isolated RNA was intact. RNA isolated after one week ~ 10 days is partially degraded (intact ~ 50%). This RNA is still a suitable substrate for Nuclease Protection Assays or RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction), both procedures being known to those skilled in the art. The data is shown in Fig. 8. Example 11

RNA isolated from amphibian, fish, insect, bacterial and plant tissues stored at 4 ° C in RNAlater ™

The Inventor studied the efficacy of RNAlater ™ in RNA preservation in Xenopus heart and liver, goldfish liver, whole beetle, whole Drosophila, E.Coli, tobacco and alfalfa. Each was placed in RNAlater ™ solution and stored at 4 ° C. After incubation for 24 hours, RNA was extracted from the samples and analyzed by denaturing electrophoresis on agarose gels as described in Example 1. All RNAs were intact. This demonstrates the efficacy of RNAlater ™ as a useful general reagent for tissues of various organisms.

Example 12

Demonstration of the suitability of RNAlater ™ RNA as a target in Northern hybridization analysis

Fresh mouse liver, kidney and spleen samples were incubated in RNAlater ™ for 24 hours at 25 ° C or 37 ° C. RNA was extracted, resolved by denaturing electrophoresis on agarose gels, transferred to a positively charged nylon membrane (Brightstar Plus ™, Ambion, Austin, Texas) and hybridized with a radioactively labeled probe of antisense RNA for β Actin. in accordance with manufacturers' recommendations (Northern Max ™ Kit, Ambion, Austin, Texas). The discrete signal corresponding to the 1.8 kb β-actin transcript detected in all samples indicates that the messenger RNA from all samples was fully intact and available for hybridization by Northern analysis.

Example 13

Convenience of RNAlater ™ RNA as a Template for RT-PCR Analysis

RNA prepared from mouse liver, spleen, kidney and testis stored in RNAlater ™ for 24 hours at 37 ° C was used as a template for RT-PCR with two pairs of PCR ™ primers for constitutively expressed genes (housekeeping genes for cyclophilin and RIG / S15) in a Multiplex RT-PCR amplification. In all cases the expected products were obtained (cyclophilin-216 bp, RIG / S15-324 bp). Therefore, the recovered RNA is suitable for RT-PCR analysis.

Example 14

Use of RNA Preservation Media to Protect Clinical Specimens

There is a growing trend of genetic analysis of human clinical samples by RT-PCR. These clinical specimens include solid tumors, isolated cells, serum, urine, blood, or stool. Solid tumor biopsies are frequently analyzed to see the expression of specific reporter genes such as p53, whose aberrant expression plays a fundamental role in various cancers. White blood cells isolated from normal or leukemic blood are frequently tested for the expression of interleukin genes. Urine, blood, serum, plasma, and feces are frequently tested for the presence of pathogenic organisms. In anticipated use, it is possible to use RNAlater ™ or other inventive RNA preservation media as a transport buffer for isolated patient specimens in clinical settings. The RNA in these samples would then be protected until the samples could be transferred to a laboratory facility where the RNA would be extracted for analysis. In anticipated use, RNAlater ™ or other inventive RNA preservation media can be used to stabilize normally unstable viral RNA (such as HIV) in blood products for later diagnosis.

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Example 15

Using RNA Preservation Media to Preserve Field Specimens

Field biologists around the world face the common problem of how to preserve samples collected in the field for later analysis in the laboratory. Investigating RNA expression is often simply avoided due to logistical difficulties related to keeping specimens frozen on dry ice or liquid nitrogen. In anticipated use, RNAlater ™ or other inventive RNA preservation media can be used as a transport buffer for specimens isolated in the field by scientists who need to preserve samples until later until later returned to the laboratory setting. An important benefit of the RNAlater ™ or other inventive RNA preservation media is that small specimens (such as microorganisms) remain intact. Therefore, complex specimens such as a population of microorganisms isolated from water samples can be conserved en masse, selected by classification in the laboratory, and then analyzed for gene expression.

Example 16

Using RNA Preservation Media as a Dissection Media

Biologists must frequently perform complicated dissections in order to isolate a specimen for RNA analysis. Time delays often mean that the RNA isolated from the sample will be of poor quality. Developmental biologists studying early embryonic development in mice must perform meticulous dissections to isolate early embryos from the decidua uterus. In another example, neuroanatomists must perform complicated dissections to remove specific parts of the brain for RNA analysis. In anticipated use, RNAlater ™ or other RNA preservation media of the invention will be a very effective preservation medium. Immersing a sample in RNAlater ™ or other inventive RNA preservation media will protect the RNA during dissection, while preserving the integrity of the sample (other reagents, such as guanidinium will cause extensive cell lysis and compromise dissection). The use of RNAlater ™ or other inventive RNA preservation media will facilitate lengthy dissection of a sample without fear of RNA in the sample degrading.

Example 17

Use of RNA Preservation Media to Preserve Specimens for Pathogenic Study

Nucleic acid analysis to detect and classify pathogenic organisms is a rapidly growing technology. In anticipated use, RNAlater ™ or other inventive RNA preservation media can be used as a transport buffer for specimens collected by sanitary inspectors in the field. Placing the samples in RNAlater ™ or other inventive RNA preservation media will preserve the RNA in the samples. The samples could then be analyzed using nucleic acid technology to detect the presence of pathogenic organisms. For example, a US Department of Agriculture inspector could collect meat samples from a slaughterhouse and place them in RNAlater ™ or other inventive RNA preservation media. The RNA of pathogenic organisms present on the surface of the sample will remain intact. The meat can later be removed from the sample, the pathogens are recovered from RNAlater ™ or other inventive RNA preservation media, and the RNA is isolated for analysis. An important feature of the RNAlater ™ or other RNA preservation media of the invention is that it is bactericidal but does not cause cell lysis. Therefore, the titer of pathogenic organisms will not change during storage of the samples or misrepresent the apparent numbers, and the samples can also be analyzed microscopically for additional information.

Example 18

Using RNA Preservation Media to Preserve Pathogens

Thousands of specimens are stored and shipped each year on dry ice to central laboratories that perform RNA analysis for pathogens. In anticipated use, RNAlater ™ or other RNA preservation media of the invention can be used as a delivery buffer, preserving RNA from infectious, disease-causing organisms. The RNAlater ™ or other inventive RNA preservation media will allow samples to be shipped at room temperature or near room temperature without fear of RNA degradation. One organization that will benefit from both the extra protection that RNAlater ™ or other inventive RNA preservation media would produce, as well as the significant savings in shipping costs, is the Center for Disease Control (CDC), which receives many animal samples for pathogenic study.

Example 19

Use of RNA Preservation Media in CCAF Classification

Classification of Cells Activated by Fluorescence (CCAF) is a method in which cells in suspension can be separated based on differences in cell surface markers. We anticipate the use of RNAlater ™

ES 2 255 293 T3 or other RNA preservation media of the invention as a solution for the suspension of cells to be processed in a CCAF equipment. In this way, the RNA of the cells will be conserved. Once the cells have been sorted, intact RNA can be isolated for analysis.

Example 20

Using RNA Preservation Media to Preserve Soil Bacteria

With the advent of RT-PCR methodology to identify bacterial species, there is a growing need for methods to isolate bacteria from soil away from soil for subsequent isolation of RNA. However, RNA from bacteria is extremely labile and degrades rapidly during the isolation protocol. An early use of RNAlater ™ or other inventive RNA preservation media is a first step in isolating RNA from soil bacteria. It can be ground dispersed in RNAlater ™ or other inventive RNA preservation media, instantly protecting the RNA within the bacteria, but keeping the bacteria intact. The soil can then be safely removed by centrifugation at a low speed, then the bacteria can be recovered by centrifugation at a higher speed. RNAlater ™ or other inventive RNA preservation media will prevent the RNA from being degraded during the isolation procedure.

Example 21

Methods for Isolating RNA Conserved in RNAlater ™ or Other Inventive RNA Preservation Media

Various methods for isolating RNA from tissue samples have been evaluated for their suitability for tissues preserved in RNA preservation media. Several such methods can be carried out with kits available from Ambion. Of course, Ambion kits are not required for the isolation of RNA from tissues preserved in RNA preservation media, and the use of other methodologies or kits to isolate RNA from tissues and cells preserved in RNA preservation media. RNA is covered by this specification. For example, any of the methods for isolating RNA, such as those of Boom et al. (Selective binding and retention of nucleic acids to a glass matrix - Qiaprep ™, Qiagen, Inc.), Chomsczynski et al. (Trizol ™, MRC), Macfarlane et al. (Castrimox 14 ™, Iowa Biotechnology, Inc.), Bugos et al. (Guanidinium: Lithium Chloride), or Auffray et al. (LiCl .: urea extraction), or kits for the practice of such methods, can be used in this regard.

The specific examples that follow describe the use of Ambion kits to isolate RNA from tissue or cell samples preserved in RNAlater ™ or other inventive RNA preservation media. It is contemplated that Ambion may choose to sell a combination kit for the preservation of RNA in tissue or cell samples, followed by subsequent isolation of the RNA from these samples.

Example 22

Isolation of cellular RNA from samples preserved in RNAlater ™ or other inventive RNA preservation media

Ambion's Totally ™ RNA Kit is a Gaunidinium / Acidic Phenol method for preparing cellular RNA.

In order to demonstrate the utility of combining RNAlater ™ or other inventive RNA preservation media and tissue sample preservation with the ToTally RNA ™ procedure, tissue samples stored in RNAlater ™ were removed and homogenized in 10 volumes. of a lytic solution of guanidinium isothiocyanate consisting of 4M guanidinium hydrochloride, 0.5% sarcosine, 25 mM sodium citrate, 0.1 M 2-mercaptoethanol. Proteins are removed by an extraction with equal volume of phenol: chloroform (1: 1), followed by centrifugation to separate the aqueous and organic phase. The aqueous phase is recovered and extracted a second time with phenol at pH 4.7. This second extraction partitions any remaining protein towards the organic phase and low pH forces any DNA towards the organic phase. The RNA remains in the aqueous phase. The aqueous phase is recovered by centrifugation. RNA is recovered from the aqueous phase by precipitation with 0.3M sodium acetate and 2.5 volumes of ethanol. The results are shown in Fig. 9.

Example 23

Isolation of cellular RNA in samples preserved in RNAlater ™ or other inventive RNA preservation media using the Ambion RNAqueous ™ kit

The RNAqueous ™ kit from Ambion is a guanidinium-containing lysis method for isolating RNA that does not require organic solvents. Instead, it relies on the selective absorption of RNA to a glass fiber filter.

In order to demonstrate the utility of combining RNAqueous ™ with RNAlater ™ or other inventive RNA preservation media, tissue samples were removed from RNAlater ™ storage, directly transferred to the lytic guanidinium isothiocyanate solution provided in RNAqueous ™ and homogenized. The homogenate was applied to a glass fiber filter and washed with various buffers, which remove proteins and DNA. The pure RNA was eluted after the water filter. The combination of RNAlater ™ or other preservation media

ES 2 255 293 T3 RNA of the invention and RNAqueous ™ has the advantage that no caustic or carcinogenic organic reagents (phenol, chloroform, ethanol, acetic acid, etc.) are used during the procedure. Fresh samples or preserved samples RNAlater ™ they were homogenized in 10 volumes of a solution consisting of 4M guanidinium hydrochloride, 1% sarcosine, 25 mM sodium citrate, 0.1 M 2-mercaptoethanol, 2% Triton X-100. The homogenate is diluted 2X and passed through a glass fiber filter. Under these conditions, the nucleic acids bind to the filter and the proteins are washed. Several sequential washes with a high concentration of salt and ethanol differentially wash the DNA, leaving the RNA bound to the filter. RNA is subsequently recovered by eluting with hot water. The results are shown in Fig. 9.

Example 24

Using Solid RNAlater ™ to Preserve Nucleic Acids in Liquid Samples

The solid components of the preferred RNA preservation composition can be dissolved directly in a liquid sample to be stabilized, added to a sample / liquid mixture, added to a sample that has been placed in a liquid or present in a collection container. before collecting a sample or sample / liquid mixture.

The solid components can be mixed in a mixer for dry products in the suitable composition such that when added to an aqueous mixture and dissolved, the preferred concentration of the salt, buffer and chelator is obtained in solution. Premeasured aliquots of the dry powdered components can be loaded into sample collection containers, and an appropriate volume of sample added. The collection container will then be shaken, dissolving the RNAlater components in the solution. This will minimize operator exposure to the sample, and will be a preferred method of stabilizing nucleic acids in biological specimens such as blood and urine. Alternatively, the solid components can be compressed into tablets and stored in bulk. The tablets will be a convenient format to store and can be added in the correct amount to a sample of any size and any type of container. This will be a preferred method for a field collection of liquid samples from sources such as water reservoirs or sewage treatment plants or in the dairy industry. The primary advantages of using an RNA preservation reagent in the dry mode will be weight savings during storage and transportation, spills of solid components are less likely, and volume savings in preserving such liquid samples with Dry reagents will minimize the final volume of the sample.

US Patent No. 5,922,591

US Patent No. 5,921,396

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