Methods and Reagents for Preserving RNA in Cell and Tissue Samples

(19) &

(11) EP 1 657 313 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12Q 1/68 (2006.01) C12N 1/04 (2006.01) 05.05.2010 Bulletin 2010/18 C12N 15/10 (2006.01)

(21) Application number: 06000216.9

(22) Date of filing: 30.07.1999

(54) Methods and reagents for preserving RNA in cell and tissue samples Methoden und Reagenzien zur Erhaltung der RNA in Zellen und Geweben Procédés et reactifs permettant de preserver l’ARN dans des prélèvements cellulaires et tissulaires

(84) Designated Contracting States: (56) References cited: AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU EP-A- 0 707 061 MC NL PT SE • DIMULESCU ET AL: "Characterization of RNA in (30) Priority: 31.07.1998 US 127435 cytologic samples preserved in a methanol- based collection solution" MOLECULAR (43) Date of publication of application: DIAGNOSIS, NAPERVILLE, IL, US, vol. 3, no. 2, 17.05.2006 Bulletin 2006/20 June 1998 (1998-06), pages 67-72, XP005235341 ISSN: 1084-8592 (62) Document number(s) of the earlier application(s) in • FOSS R D ET AL: "Effects of Fixative and Fixation accordance with Art. 76 EPC: Time on the Extraction and Polymerase Chain 99940837.0 / 1 019 545 Reaction Amplification of RNA From Paraffin- embedded Tissue Comparison of Two (73) Proprietor: Ambion, Inc. Housekeeping Gene mRNA Controls" Austin, TX 78704 (US) DIAGNOSTIC MOLECULAR PATHOLOGY,US, NEW YORK, NY, vol. 3, no. 3, 1994, page 148-155, (72) Inventor: Lader, Eric S. XP000575426 Boyds, MD 20841 (US) • CHOMCZYNSKI P ET AL: "SINGLE-STEP METHOD OF RNA ISOLATION BY ACID (74) Representative: Casalonga, Axel et al GUANIDINIUM THIOCYANATE-PHENOL- Casalonga & Partners CHLOROFORM EXTRACTION" ANALYTICAL Bayerstrasse 71/73 BIOCHEMISTRY,US,ORLANDO, FL, vol. 162, no. 80335 München (DE) 1, 1987, page 156-159, XP000608462 ISSN: 0003-2697 • CHOMCZYNSKI P: "A REAGENT FOR THE SINGLE-STEP SIMULTANEOUS ISOLATION OF RNA, DNA AND PROTEINS FROM CELL AND TISSUE SAMPLES" BIOTECHNIQUES,US, EATON PUBLISHING, NATICK, vol. 15, no. 3, 1993, page 532-536, XP000604677 ISSN: 0736-6205 • CATHALA G ET AL: "A method for isolation of intact, translationally active ribonucleic acid" DNA,US,NEW YORK, NY, vol. 2, no. 4, 1983, page 329-335, XP002095897

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 1 657 313 B1

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Description mortar and pestle is extremely laborious. [0005] Quick freezing is even less convenient outside BACKGROUND OF THE INVENTION of the laboratory environment, but is still considered a necessity by those in the field. Scientists in the field col- 1. Field of the Invention 5 lecting specimens for analysis do not have access to a high-speed homogenizer. They are forced to carry a sup- [0001] The present invention relates to the field of mo- ply of liquid nitrogen or dry ice large enough to store sam- lecular biology and provides a novel method and reagent ples until they can be transferred to an ultra- low temper- for preserving and protecting the ribonucleic acid (RNA) ature freezer. Similarly, RNA extracted from human bi- content of tissue or cell samples from degradation prior 10 opsy samples is usually partly or mostly degraded be- to RNA isolation. cause pathologists do not routinely flash freeze specimens to preserve RNA. 2. Description of Related Art [0006] There have been attempts to isolate RNA from archival samples that have not been prepared by the [0002] Obtaining high quality, intact RNA is the first 15 flash-freezing methodology. For example, Esser et al., and often the most critical step in performing many fun- 1995 claim the isolation of full length RNA from cells fixed damental molecular biology experiments. Intact RNA is with 5% acetic acid, 95% ethanol, with RNase inhibitors. required for quantitative and qualitative analysis of RNA However, in this paper, isolated cells in suspension were expression by Northern blot hybridization, nuclease pro- fixed in acetic acid/ethanol solution at -20 °C and then tection assays, and RT-PCR. 20 held at 4 °C for a relatively short time. Unfortunately, test- [0003] There are many published reports which de- ing by the Inventor has shown that the Esser et al. 95% scribe methods to isolate intact RNA from fresh (or quick ethanol/5% acetic acid solution does not meet the per- frozen) cells or tissues. Most of these techniques utilize formance standards required by the present invention. a rapid cell disruption step in which the tissue is dispersed RNA recovered from both tissue samples and spleen in a powerful protein denaturation solution containing a 25 cells in suspension kept at 4°C for 20 hours appeared chaotropic agent (e.g., guanidinium or lithium salt). This partially degraded, while RNA isolated from tissues rapid disruption of cell membranes and inactivation of stored at ambient temperature was completely degrad- endogenous ribonuclease is critical to prevent the RNA ed. Experiments reported in Esser et al. show that the from being degraded. method results in loss of RNA, due to leakage from the [0004] To obtain high quality RNA it is necessary to 30 cells caused by ethanol. Using that method, 70% of the minimize the activity of RNase liberated during cell lysis RNA is lost immediately upon fixation, and after 1 hour, and to prevent RNA degradation from other sources. This 80% of the RNA is gone. Further, in a test where tissue is normally accomplished by using isolation methods that samples and spleen cells were stored in the 95% ethanol/ disrupt tissues and inactivate or inhibit RNases simulta- 5% acetic acid solution at 25 °C overnight, the RNA of neously. For specimens low in endogenous ribonucle- 35 both the cell and tissue samples was completely degrad- ase, isolation protocols commonly use extraction buffers ed. Data is shown in FIG. 1. containing detergents to solubilize membranes, and in- [0007] The use of high purity, intact RNA is fundamen- hibitorsof RNase such asplacental ribonuclease inhibitor tal for performing various molecular biological assays or vanadyl-ribonucleoside complexes. RNA isolation and experiments such as Northern blot hybridization, nu- from more challenging samples, such as intact tissues 40 clease protection assays, RT-PCR and medical diagno- or cells high in endogenous ribonuclease, requires a sis. The intrinsic instability of RNA and the presence of more aggressive approach. In these cases, the tissue or RNases in samples makes the isolation of intact RNA a cells are quickly homogenized in a powerful protein de- difficult procedure. Further, the isolation and assay of naturant (usually guanidinium isothiocyanate), to irre- RNA-containing samples is typically time consuming and versibly inactivate nucleases and solubilize cell mem- 45 tedious. The contamination of a molecular biology labo- branes. If a tissue sample can not be promptly homoge- ratory with RNases due to human error can have cata- nized, it must be rapidly frozen by immersion in liquid strophic results. Thus, there is an ongoing need to de- nitrogen, and stored at -80°C. Samples frozen in this velop improved techniques, to make RNA isolation and manner must never be thawed prior to RNA isolation or assay methods more sensitive, more specific, faster, the RNA will be rapidly degraded by RNase liberated50 easier to use and less susceptible to human error and during the cell lysis thatoccurs during freezing. Thetissue handling. It would therefore be advantageous in many must be immersed in a pool of liquid nitrogen and ground instances, for research facilities to use automated RNA to a fine powder using mortar and pestle. Once pow- preservation protocol. For example, the present inven- dered, the still-frozen tissue is homogenized in RNA ex- tion, could be combined with rapid RNAassay techniques traction buffer. In the laboratory, quick freezing of sam- 55 or integrated nucleic acid diagnostic devices (U. S. Pat- ples in order to delay RNA extraction carries the penalty ent 5,726,012, U.S. Patent 5,922,591, ) for efficient, au- of a substantial increase in hands-on processing time. tomated RNA preservation and analysis. Processing multiple samples with liquid nitrogen and [0008] U. S. Patent No. 5,256,571 reports a cell pre-

2 3 EP 1 657 313 B1 4 servative solution comprising a water-miscible alcohol in (3M). The authors theorize that the combination of the an amount sufficient to fix mammalian cells, an anti- neutral pH and the high salt concentration forces a re- clumping agent and a buffering agent. At least one paper, folding of the protein into an alternate, highly active con- Dimulescu et al., reports the apparent use of this fixative figuration. However, the Allewell et al. group were exam- to preserve cervical cancer cells and cord blood lym- 5 ining the activity of pure RNase A in solution, rather than phocytes prior to RNA isolation. in a cellular sample containing many RNases. [0009] A large body of literature suggests that ethanol [0012] In view of the above, there is a need for methods and acetone combinations are the best known fixatives and reagents that allow one to preserve and recover high for future recovery of nucleic acids from archival tissue. quality, intact RNA from tissue samples stored at near Yet, in view of the studies of the inventors, such ethanol/ 10 ambient or ambient temperature. acetone mixture does not provide all of the desired char- acteristics of an RNA preservation medium. The mixtures SUMMARY OF THE INVENTION do not protect RNA at ambient temperature, does not allow for the preservation of RNA in solid, multi- cell sam- [0013] The present invention relates to a novel method ples, and are also flammable, which makes it intrinsically 15 for preserving the RNA in tissue fragments at tempera- less attractive as a general use reagent. tures above the freezing point of the preservatives for [0010] Some peripherally related art exists that deals extended periods of time, including days to months, prior with aspects of preserving or recovering RNA from fixed to RNA isolation. There is no prior report disclosing any or preserved tissue samples. These reports include nu- reagent or method similar to that described in this appli- merous evaluations of the suitability of histological fixa- 20 cation. This breakthrough alleviates the necessity of ei- tives to maximize the signal obtained by in situ hybridi- ther immediately processing samples to extract RNA, or zation to detect (not recover) RNA in tissue samples (for the restriction of only isolating tissue at sites which have example U.S. Patent Nos. 5,196,182 and 5,260,048). a supply of liquid nitrogen or dry ice. Other reports detail methods to recover fragmented RNA [0014] The present application relates to compositions from fixed tissues for limited molecular analysis 25 by of RNA preservation media and methods of preserving PCR™ (Koopman et al., Foss et al., Stanta et al., Houze RNA comprising: (1) obtaining an RNA-containing sam- et al.). To recover this fragmented RNA, samples are ple; and (2) treating the sample with an RNA preservation typically treated with proteinase K to degrade the struc- medium that infiltrates the sample, and protects the RNA tural components of the tissue, then the RNA is extracted from nucleases. In a preferred embodiment, the RNA with a guanidinium-based solution. The RNA recovered 30 preservation medium brings about the precipitation the from fixed tissue is of extremely poor quality, averaging RNA in the sample along with cellular protein in the sam- in size of about 200 bases (Stanta 1991). This is probably ple. This co- precipitation of the RNA and cellular proteins due to a number of factors including the action of endog- is believed to render the RNA inaccessible to nucleases enous RNase and cross-linking of the RNA in the intra- via physical means, while the action of the RNA preser- cellular matrix during fixation. Since the RNA is mostly 35 vation medium simultaneously inactivates or inhibits the degraded, it can not be used for northern analysis or nu- action of the nucleases. clease protection assays. It can be used in RT- PCR, but [0015] The RNA preservation medium comprises a only for amplification of very small fragments. salt that precipitates the RNA in the sample along with [0011] The use of ammonium sulfate to precipitate pro- the cellular protein, wherein, the salt is ammonium sul- teins out of solution is known, but the use of ammonium 40 fate. sulfate to preserve RNA does not, to the Inventor’s knowl- [0016] In RNA preservation media comprising ammo- edge, appear in the art. Two reports describe the use of nium sulfate, the salt is present in a concentration suffi- ammonium sulfate to investigate the folding and activity cient to precipitate the RNA in the sample along with the of mammalian ribonuclease A (Allewell et al. and Lin et cellular protein. The salt is present in a concentration al.). Allewell et al. investigated the effects of ammonium 45 between 30 g/100 ml and 40 g/100 ml. Specifically, salt sulfate on the folding and activity of RNase A. At pH 5.5, concentrations of 30, 35, 40 g/ 100 ml may be used, and the activity of ribonuclease A is suppressed to approxi- the concentration may be a range defined between any mately 10% of the untreated control level across a broad two of these concentrations. range of ammonium sulfate concentration. This suppres- [0017] Of course, during use, some dilution of the salt sion of activity was expected by the authors. It appears 50 concentration may occur due to, for example, liquid in to be due to a salt-induced denaturation of the protein. the sample. Therefore, these salt concentrations may be Unfortunately, even 10% RNase activity would substan- higher than the final salt concentrations obtained in some tially degrade the RNA of a sample over time. Therefore, uses. Further, it is disclosed herein that amounts of salt this inhibition is not sufficient to protect RNA in many above the saturating concentration may be used. There applications. When the ammonium sulfate is at pH 7.0, 55 may be salt that is not in solution in the RNA preservation the activity of RNase A is suppressed at low concentra- medium. This should not affect the RNA preservation tions as expected, but unexpectedly rises to 110% of the abilities of the media. In fact, media having more than a level of the untreated control at higher concentrations saturating concentration of a salt may have some utility

3 5 EP 1 657 313 B1 6 in applications where the media is added to a liquid sam- acid. These additional potential components can precip- ple. In such cases, upon addition to the liquid sample, itate proteins in preserved cells and thereby protect RNA. salt which is not in solution prior to addition, may become However, these additional potential components are not soluble due to the increase in liquid volume. Thus, the salts. It is anticipated that in some embodiments, one will final concentration of a salt can be at a level higher than 5 use these organic solvents in combination with a con- that possible if a preservation medium containing a sat- centration of salt to obtain one of the inventive RNA pres- urating or less than saturating salt concentration were ervation media described herein. For example, it is an- used. ticipated that a combination of one or more of these or- [0018] Also disclosed herein is that other salts or com- ganic solvents in conjunction with less than 20 g /100 ml pounds will also be useful in protecting RNA in tissue 10 salt will accomplish the goals of the invention. samples and cell samples, for reasons as follows. The [0020] The RNA preservation medium may comprise solubility of individual proteins depends greatly on the a chelator of divalent cations, for example EDTA. pH and salt concentration of the aqueous environment. [0021] Typically, the RNA preservation medium com- Virtually all proteins are insoluble in pure water. As the prises a buffer so that a constant pH can be maintained. ionic strength of the medium increases, proteins become 15 For example, the buffer can be sodium citrate, sodium more soluble. This is known as "salting in" of proteins. acetate, potassium citrate, or potassium acetate. In a Above some ionic strength, the solubility of protein de- presently preferred commercial embodiment, the buffer creases. The precise conditions at which this occurs is is sodium acetate. Typically, the RNA preservation me- unique for each protein/ salt combination. In fact, at some dium has a pH of between 4 and 8. In presently preferred salt concentrations, one protein may be completely in- 20 commercial embodiments, the pH is 5.2. soluble while another is at its solubility maximum. This [0022] The sample preserved in the RNA preservation phenomenon is known as "salting out." Some salts have media may be any of a number of types of samples. For a much more dramatic salting out effect at high concen- example, the sample may be a suspension of cells, such - - 2- trations than others (e.g., NO3

4 7 EP 1 657 313 B1 8 infiltrates the sample and protects or partitions the RNA still other embodiments, the RNA preservation composi- from nucleases; and (2) a reagent for performing an RNA tion comprises a buffer, wherein said buffer has a pH extraction from the sample. The reagent for performing between 4 and 8. an RNA extraction may be a reagent for performing RNA [0030] The solid components of the present invention extraction without organic solvents. Further, the reagent 5 (e.g., ammonium sulfate salt, buffers, and shelters), can for performing an RNA extraction may be a reagent for be prepared to yield the desired final component concen- performing a guanidinium-based RNA extraction. Alter- trations in solution when added to an aqueous sample. natively, the reagent for performing an RNA extraction is The solid components can further be provided as pow- a reagent for performing a lithium chloride-based RNA ders, tablets, pills or other suitable formulations that pro- extraction. 10 vide the desired properties of an RNA preservation com- [0026] In other embodiments of the present invention, position. Solid components can be directly added to a a method of preserving RNA will comprise obtaining an sample, added to a sample/liquid mixture, or present in RNA-containing sample, providing the ammonium sul- a collection vessel prior to collection of a sample or sam- fate salt and admixing the sample and the salt in a liquid ple/liquid mixture. The addition of excipients and bulking to form an RNA preservation composition that infiltrates 15 agents such as mannitol, lactose, starch, cellulose, and the sample and protects the RNA from nucleases. In one the like, to provide desired solid charctersitics i.e.( , im- embodiment, the sample is comprised in the liquid prior proved solubility, storage stability, particle dispersion) to admixing the sample with the ammonium sulfate. In are also considered in the formulation of powders, tablets another embodiment, the ammonium sulfate is in a solid and pills. The solid components of the present invention form prior to admixing with the sample and the liquid. In 20 can be added prior to sample collection, after sample yet another embodiment, the ammonium sulfate is com- collection or any combination thereof. prised in the liquid prior to admixing the sample with the [0031] In one embodiment of the invention, pre-meas- ammonium sulfate. ured aliquots of a solid or liquid RNA preservation com- [0027] In one embodiment of the invention, the sample position can be loaded into sample collection vessels, is a blood cell and the liquid is blood serum. In another 25 and an appropriate volume of a RNA- containing sample embodiment the sample is urine. In other embodiments, added. The collection vessel would then be agitated, dis- the liquid is water. In yet other embodiments, the liquid solving any solid components of the RNA preservation is a buffer. composition, minimizing operator exposure to an RNA [0028] In certain embodiments, RNA preservation sample. For example, a solid RNA preservation compo- compositions comprise obtaining an RNA-containing 30 sition is ammonium sulfate salt. Thus, in particular em- sample, providing the salt ammonium sulfate and admix- bodiments of the invention, stabilizing RNA in a biological ing the sample and the salt in a liquid. The liquid can be specimen such as blood or urine as described above is a component of the sample, or added to the sample and contemplated. salt. The ammonium sulfate is typically present in a con- [0032] In one example, a vial for collecting specimen centration sufficient to precipitate the RNA in the sample 35 such as urine or blood could be supplied with pre-meas- along with the cellular protein. The ammonium sulfate is ured aliquots of a RNA preservation composition. Imme- typically added so as to result in a final concentration in diately following collection of said specimen, the vial solution between 30 g/100 ml and 40 g/100 ml. In some could be agitated, admixing the RNA-containing speci- cases, it will be efficient to add a very concentrated am- men (i.e., sample) with the RNA preservation composi- monium sulfate salt, a saturated salt or a supersaturated 40 tion. A blood collecting vacuum vial comprising an out- salt to a liquid sample. Adding salt that is likely to result wardly projecting needle is contemplated for us in the in a greater than saturating concentration of salt in the present invention (U.S. Patent 5,090,420,) for rapid col- final liquid sample has the advantages of quickly allowing lection and mixing of RNA-containing samples. In one RNA preserving concentrations to be reached and avoid- embodiment, automated RNA preservation is contem- ing the need to carefully consider the amount of salt need- 45 plated. Automated RNA preservation methods would be ed to reach a specific concentration in a given sample. less tedious, faster, easier to use and less susceptible Further, any salt that does not go into solution, will not to human error and handling. affect the RNA preserving properties of the composition. [0033] Also contemplated for use with pre-measured Specifically, final ammonium sulfate concentrations of aliquots of a RNA preservation composition of the 30, 35, 40 g/100 ml may be used, and the concentration 50 present invention are clinical specimen collection kits may be a range defined between any two of these con- adapted for shipment by mail (U.S. Patent 5,921,396,). centrations. In some cases, a liquid containing more than RNA-containing samples could be collected (e.g., away a saturating concentration of ammonium sulfate may be from a laboratory facility) and mixed with pre-measured employed, with the expectation that additional volume of aliquots of a RNA preservation composition provided in liquid in the sample will result in dissolution of salt con- 55 the vial, preserving RNA for shipment to a suitable RNA centration. analysis site. [0029] In another embodiment, the RNA preservation [0034] Alternatively, a RNA preservation composition composition comprises a chelator of divalent cations. In can be pressed into a tablet or pill and stored in bulk.

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Tablets would be a convenient composition for storage in the inventive buffers to be degraded. Allewellet al. and could be added in the correct quantity to a sample reports that at pH 5 RNase is more active than normal. of any size in any container type. Thus, in other embod- Thus, if RNAlater™ acted by the same mechanism, one iments of the present invention, RNA preservation com- would expect a boosting of the activity of RNase. Obvi- ponents in the form of tablets or pills are contemplated 5 ously, this would limit the ability of the RNA preservation for field collection of samples from such sources as water media to preserve RNA. Based on the observation that reservoirs, sewage plants or the dairy industry. In certain such degradation does not occur, the present invention instances, RNA preservation media comprising prede- functions by a different mechanism than that described termined final salt concentrations or supersaturated salts by Allewell et al. could be supplied as packets. The RNA preservation am- 10 [0040] The term "RNAlater™" is a trademark of Ambi- monium sulfate salt or supersaturated ammonium sulfate on, Inc., for certain commercial formulations of the RNA salt packets would be especially useful in field studies, preservation media disclosed herein. In general, the term as there may be limited space or resources for analytical "RNAlater™" is employed to denote the formulation dis- equipment. For example, packets could be supplied, as closed in Example 2, which is composed of 25 mM So- pre-measured and packaged as aliquots for a 1 mL, 5 15 dium Citrate, 10 mM EDTA, 70 g ammonium sulfate/100 mL, 10 mL, sample. Of course, any size packet, to ac- ml solution, pH 5.2. This reagent functions by rapidly in- commodate a variety of salt quantities could be provided filtrating cells with a high concentration of ammonium sul- as either an anhydrous powder, hydrous powder, or a fate, causing a mass precipitation of cellular proteins. powder and liquid packaged individually or any combi- Importantly, cellular structure remains intact. The advan- nation thereof. Thus, to preserve RNA in a sample, one 20 tage of this is that cells can be preserved and still iden- would simply add the packet contents to a sample and tified histologically. mix. [0041] Following long-standing patent law, the words [0035] Certain advantages of using a solid component "a" and "an," when used in conjunction with the word RNA preservation composition would be weight savings "comprising" in the claims or specification, denotes one in storage and transport, spills of solid components are 25 or more. less likely and reduced volume savings (i.e., preserving samples with dry reagents would minimize the final vol- BRIEF DESCRIPTION OF THE DRAWINGS ume of the sample). [0036] In other embodiments of the invention, RNA [0042] The following drawings form part of the present preservation compositions further comprise isolating the 30 specification and are included to further demonstrate cer- preserved RNA, wherein the RNA is isolated at a tem- tain aspects of the present invention. The invention may perature that is greater than -20°C. In other embodi- be better understood by reference to one or more of these ments, the sample is stored prior to the isolation of the drawings in combination with the detailed description of RNA, wherein the sample is stored at temperatures specific embodiments presented herein. greater than 0°C. 35 [0037] In particular embodiments, a composition used FIG. 1 -- Alcohol and acetone are unsuitable for pre- herein comprises an RNA-containing sample, a liquid, serving cellular samples. and ammonium sulfate salt in a concentration between 30 and 40 g/ 100 ml. In another embodiment, the compo- [0043] Panel A at 4°C (lanes 1-5:ethanol, acetic etha- sition comprises a buffer, having a pH between 4 and 8. 40 nol, acetone, acetic acetone, RNAlater™). Panel B In yet another embodiment, the composition comprises shows identical lanes at 37°C. RNA isolated from mouse a chelator of divalent cations. liver stored overnight at 4°C (lanes 1-5) or 37°C (lanes [0038] The inventor’s research indicates that 3M am- 6-10). Lanes 1 and 6, storage in ethanol. Lanes 2 and 7, monium sulfate at pH 7.0 is quite effective at preserving storage in acidified ethanol pH 4.0. Lanes 3 and 8, stor- intact RNA in intact tissue samples at all but extreme45 age in acetone. Lanes 4 and 9, storage in acidified ace- temperatures (37°C-42°C). It is proposed that upon ap- tone pH 4.0. Lanes 5 and 10, storage in RNAlater™. plication to the sample, the ammonium sulfate diffuses into the tissue and cells and causes cellular proteins to FIG. 2 -- RNA in fresh tissue samples is labile. precipitate (probably along with RNA which is intimately associated with many different proteins in vivo) in pro- 50 [0044] Fresh mouse liver (lanes 1 and 2), testis (lanes tected complexes. In addition, RNase, which is localized 3 and 4), and spleen (lanes 5 and 6) samples were placed in cytoplasmic vesicles, may also be precipitated and ren- either in RNAlater™ (lanes 2,4, and 6) (25 mM Sodium dered inaccessible to cellular RNA. Citrate, 10 mM EDTA, 70 gm Ammonium Sulfate/ 100 ml [0039] It is believed that the mode of action of the solution, pH 5.0) or in a 4 molar guanidinium isothiocy- claimed invention and the mode of action of the Allewell 55 anate (GITC) based RNA extraction solution (lanes 1, 3, et al. mixture are different. If the mode of action of the and 5) at 4°C for 12 hours. RNA was extracted and an- inventive solutions were the same as seen by Allewell et alyzed by gel electrophoresis. Intact RNA is observed al.one would expect the RNA isolated from tissues stored only in lanes corresponding to tissues preserved in

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RNAlater™ (lanes 2, 4, and 6). FIG. 8 -- RNA isolated from mammalian tissue stored at extreme temperatures (37°C) in RNAlater™. FIG. 3 -- Defining the effective concentration range of Ammonium Sulfate that protects RNA in tissue. [0050] Fresh mouse liver, kidney, and spleen (lanes 5 1-3) were placed in RNAlater™ solution and stored at [0045] Fragments of freshly isolated mouse liver was 37°C. After incubation for three days, RNA was extracted placed in RNAlater™ with various amounts of Ammoni- from the tissue samples and analyzed by denaturing aga- um Sulfate. Samples contained 0, 10%, 20%, 30%, 40%, rose gel electrophoresis 50%, or 70% Ammonium Sulfate (lanes 1-7 respectively). After incubation at 4°C for 24 hours, RNA was extracted 10 FIG. 9 -- Using the Ambion to make RNA from tissues from the sample and analyzed by denaturing agarose gel preserved in RNAlater™. electrophoresis. [0051] Lanes 1-3 are liver, heart, and kidney, ToTally FIG. 4 -- Enhancing the potency of RN Alater™ at RNAT™, lanes 4-6 are RNAqueous™. Fresh mouse liver extreme temperatures by optimizing both pH and 15 (lanes I and 4), heart (lanes 2 and 5), and kidney (lanes Ammonium Sulfate concentrations. 3 and 6) samples were placed in RNA later™ and stored overnight at 4°C. RNA was isolated from size matched [0046] The effects of pH and Ammonium Sulfate at ex- samples using either the Ambion ToTally RNA™ kit treme temperatures was assessed. Fresh mouse liver (lanes 1-3) or the Ambion RNAqueous™ kit (lanes 4-6). was stored at room temperature or 37°C for 24 hours in 20 Lane 1 - molecular weight marker. 4 formulations of RNAlater™ solution. Lane 1: pH 7.0, 70 g/100ml Ammonium Sulfate, Lane 2: pH 5.0, 55g/100 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS ml Ammonium Sulfate, Lane 3: The original RNAlater™ formulation (which contained 55 g/100 ml Ammonium [0052] The present invention relates to the field of mo- Sulfate at pH 7.0), Lane 4:pH 5.0 and 70g/100 ml Am- 25 lecular biology and provides novel methods and reagents monium Sulfate concentration for preserving and protecting the ribonucleic acid (RNA) content of RNA-containing samples fromdegradation pri- FIG. 5 -- The specificity of Ammonium Sulfate on the or to RNA isolation. Strikingly, this is accomplished with- effectiveness of RNAlater™. out ultra-low temperature storage or disruption of the 30 samples. For example, isolated human biopsy tissue can [0047] Panel A and B: Fresh liver samples were incu- be placed in a RNA preservation medium and stored re- bated in 5 buffers for 3 days at 25°C (panel A) and 37°C frigerated for an extended period of time, until the re- (panel B), lane 1; RNA later™ with ammonium carbonate, searcher has time to extract RNA for analysis. lane 2 ; with ammonium chloride, lane 3; with potassium [0053] The following examples illustrate the utility of sulfate, lane 4; with magnesium sulfate lane 5; Ammoni- 35 the present invention. All examples make use of fresh um Sulfate. Panel C: Fresh liver incubated overnight at animal or plant tissues, living cells in suspension or other 4°C in RNAlater™ containing: lane 1, Cesium Chloride; samples containing RNA. The methods and composi- lane 2, Cesium Sulfate; lane 3, Ammonium Sulfate. tions are applicable for the preservation of RNA from a broad range of bacterial, plant, and animal species, in- FIG. 6 -- RNA isolated from mammalian tissues 40 cluding humans. RNA samples recovered in these ex- stored at 4°C in RNAlater™. periments were analyzed by formaldehyde/agarose gel electrophoresis, staining with ethidium bromide, and il- [0048] Fresh mouse brain, heart, kidney, liver, and lumination with 300 nm ultraviolet light, as described in spleen (lanes 1-5) were placed in RNAlater™ solution Molecular Cloning, A Laboratory Manual. (Maniatis,et and stored at 4°C. After incubation of one week (panel 45 al.). A), 2 weeks (panel B), and 4 weeks (panel C), RNA was [0054] The examples are included to demonstrate pre- extracted from the tissue samples and analyzed by de- ferred embodiments of the invention. It should be appre- naturing agarose gel electrophoresis. ciated by those of skill in the art that the techniques disclosed in the examples which follow represent tech- FIG. 7 -- RNA isolated from mammalian tissues 50 niques discovered by the inventor to function well in the stored at ambient temperature (25°C) in RNAlater™. practice of the invention, and thus can be considered to constitute preferred modes for its practice. [0049] Fresh mouse brain, heart, kidney, liver, and spleen (lanes 1-5) were placed in RNAlater™ solution and stored at 25°C (ambient temperature). After incuba- 55 tion of two weeks (panel A) or 4 weeks (Panel B), RNA was extracted from the tissue samples and analyzed by denaturing agarose gel electrophoresis.

7 13 EP 1 657 313 B1 14

Example 1 [0061] In a beaker, combine 40 ml 0.5 M EDTA, 25 ml 1M Sodium Citrate, 700 gm Ammonium Sulfate and 935 Criteria for Analysis of RNA to Determine if it is "In- ml of sterile distilled water, stir on a hot plate stirrer on tact" low heat until the Ammonium Sulfate is completely dis- 5 solved. Allow to cool, adjust the pH of the solution to [0055] The Inventor routinely performs assays on RNA pH5.2 using 1M H2SO4. Transfer to a screw top bottle designed to assess the intactness of such samples. This and store either at room temperature or refrigerated. criterion was used in the examples that follow to objec- tively gauge the quality of the RNA recovered from tis- Example 3 sues preserved in RNA later™, other inventive RNA pres- 10 ervation media, or other solutions. Exemplary General Manner of Preserving Tissue in [0056] RNA is analyzed by electrophoresis on formal- RNA Preservation Media dehyde agarose gel using the basic protocol described in "Molecular Cloning, a Laboratory Manual" (Maniatis, [0062] Tissue samples to be stored in RNAlater™ or Fritsch, Sambrook, eds., Cold Spring Harbor Press). In 15 other inventive RNA preservation media should be ex- order to visualize the RNA in the gel, the intercalating cised from the source as quickly as possible and placed dye Ethidium Bromide is added to the samples. Ethidium in RNAlater™ or other inventive RNA preservation me- Bromide, when intercalated into nucleic acid, fluoresces dia. Some tissue samples may have a protective mem- under ultraviolet light, permitting visualization of the nu- brane or other barrier that would impede the rapid infu- cleic acid. Intact RNA appears as a broad smear of het- 20 sion of RNAlater™ or other inventive RNA preservation erogeneous mRNA (from 0.5 kilobase up to ~10 kilo- media, such as a waxy coating on a leaf or the capsule base), with two very prominent, discrete bands, (28S and of a kidney or testis. These protective barriers should be 18S ribosomal RNA), superimposed on the background disrupted to allow rapid infiltration of the RNAlater™ or smear in a 2:1 ratio. There should be very little evidence other inventive RNA preservation media into the sample. of discrete bands intermediate in size between 18S and 25 In addition, large samples should be dissected into small- 28S RNA. Partially degraded RNA is characterized by a er fragments to maximize diffusion. As a general guide- loss of high molecular heterogeneous RNA, multiple line, sample thickness would be limited to 0.5 cM in at smaller ribosomal RNA cleavage products, and a devia- least two dimensions. Samples that consist of cells in tion from the 2: 1 ratio of 28S to 18S (28S is more sensitive suspension should be concentrated into a small volume to degradation). Severely degraded RNA will have 28S: 30 by gentle centrifugation at a g-force sufficient to pellet 18S ratios less than 1: 1, and exhibit a smear of degraded the cells without damage, resuspended in a minimal vol- ribosomal RNA from ~2 kilobase down to <0.1 kilobase. ume of the removed supernatant, then mixed with 5 vol- [0057] "Partially degraded," as used in this specifica- umes of RNA later™ or other inventive RNA preservation tion means that the ratio of 28S:18S rRNA is aberrant media (v/v). If concentration is not possible, the cell sus- and may be as low as 1:1, but the rRNA bands are still 35 pension should be diluted in 10 volumes of RNAlater™ distinct. or other inventive RNA preservation media. Alternatively, [0058] "Mostly degraded," as used in this specification any other volume of liquid, essential media or amount of means that the ratio of 28S:18S is below 1:1. 28S may a solid RNA preservation composition which results in be barely visible, but there is still nucleic acid in a smear the preservation of RNA scan be used. Since the buffer extending from the approximate position of 28S rRNA 40 will not disrupt the cells, concentration by centrifugation down. can be performed later. [0059] "Completely degraded," and "degraded," as [0063] Samples that are to be stored for 1 week or less used in this specification means that the only RNA may be stored at ambient temperature (25°C). For ex- present is in a low molecular weight smear below the tended stability, samples should be stored refrigerated. normal position of 18S rRNA 45 For permanent archival storage (months - years), samples may be stored in a standard freezer (- 20°C). To iso- Example 2 late RNA from treated samples, tissue samples should be transferred directly into tissue extraction buffer. Cells Preparation of an Exemplary RNAlater™ RNA Pres- in suspension must be pelleted by centrifugation, then ervation Medium 50 resuspended in RNA extraction buffer and processed.

[0060] The description in this example provides one Example 4 manner in which RNA later™ can be prepared. First, one should prepare or obtain the following stock solutions RNA in fresh tissue samples is labile and reagents: 0.5 M EDTA disodium, dihydrate(18.61 55 g/100 ml, pH to 8.0 with NaOH while stirring); 1M Sodium [0064] Fresh mouse liver, testis, and spleen samples Citrate trisodium salt, dihydrate (29.4 g/100 ml, stir to (~0.5 cm3) were placed either in RNAlater™ or in a 4 dissolve); Ammonium Sulfate, powdered; Sterile water. molar guanidinium isothiocyanate (GITC) based RNA ex-

8 15 EP 1 657 313 B1 16 traction solution, or water at 4°C for 12 hours. This gua- Example 6 nidinium solution is a typical RNA extraction buffer (and found in Ambion’s commercially available ToTally RNA™ Enhancing the potency of RNA preservation media and RNAqueous™ kits). GITC is a powerful chaotropic at extreme temperatures by optimizing both pH and agent used either alone or in conjunction with other rea- 5 Ammonium Sulfate concentrations gents in virtually all RNA isolation protocols. After an overnight incubation, RNA was extracted from the tissue [0068] The effect of pH and ammonium sulfate at ex- samples and analyzed by denaturing agarose gel elec- treme temperatures was assessed. Fresh mouse liver trophoresis. Intact RNA was observed only in lanes cor- was placed in 4 formulations of test RNA preservation responding to tissues preserved in RN Alater™. RNA ex- 10 media solution and stored at room temperature, or 37°C, tracted from samples stored in GITC was badly degrad- for 24 hours. RNA was extracted from the tissue samples ed. Thus, GITC does not preserve the RNA in intacttissue and analyzed by denaturing agarose gel electrophoresis. samples. Animal tissues stored in water or biological buff- The formulation which contained 55g/100ml ammonium ers such as normal saline yield no measurable RNA after sulfate at pH 7.0 was effective at ambient temperature, an identical overnight incubation. Data is shown in FIG. 2. 15 but did not fully protect the RNA at 37°C. A combination of low pH (5.0) and a higher ammonium sulfate concen- Example 5 tration (70 g/100 ml) was much more effective at high temperatures than the original formulation and yielded Determining the effective concentration of Ammo- intact RNA after 3 days at 37°C. Neither modification nium Sulfate that protects RNA in tissue 20 alone(low pH or higher ammonium sulfate concentration) appreciably enhanced the stability of the RNA above that [0065] Freshly isolated mouse liver was placed in a provided by the original formulation. Data is shown in variety of putative RNA preservation media with various FIG. 4. concentrationsof ammonium sulfate. Samples contained 0, 10%, 20%, 30%, 40%, 50%, or 70% Ammonium Sul- 25 Example 7 fate. After incubation at 4°C for 24 hours, RNA was extracted from the sample and analyzed by denaturing aga- The specificity of Ammonium Sulfate on the effec- rose gel electrophoresis. tiveness of RNA preservation media [0066] In the sample with no ammonium sulfate, all that is observed is a low molecular weight smear. No30 [0069] Four additional formulations of test RNA pres- specific banding of ribosomal RNA can be observed. At ervation media were produced. The ammonium sulfate 10% ammonium sulfate, slight evidence of rRNA bands was replaced with saturating amounts of ammonium car- can be observed. At 20% ammonium sulfate, the 18S bonate, ammonium chloride, potassium sulfate, or mag- band is more obvious, and there is more of a smear of nesium sulfate. Fresh liver samples were incubated in degraded 28S rRNA extending down from the anticipated 35 these buffers for 3 days at 25°C and 37°C. RNA was position of 28S rRNA. No specific 28S band is observed. extracted and analyzed by denaturing agarose gel elec- At 30% ammonium sulfate, both 28S and 18S rRNA trophoresis. Only the ammonium sulfate control prevent- bands are visible, however there is extensive degrada- ed the RNA from being degraded. Maximum protection tion (based on the aberrant ratio of 28S:18S, and numer- appears to require high concentrations of both ammoni- ous smaller bands observed. The sample with 40% am- 40 um and sulfate ions. Ammonium sulfate is probably the monium sulfate appears mostly intact, and the yield is 10 most effective of the salts tested because it is far more fold higher than at 30%. While these experiments sug- soluble in water than these other salts, enabling formu- gest a minimum requirement for 30-40% ammonium sul- lations with very high salt content. This hypothesis was fate for protection of tissue RNA, the effective concen- challenged by evaluating two esoteric (and more expen- tration will depend on the type of tissue, the size of the 45 sive) salts; cesium sulfate (which is quite soluble - satu- tissue fragment, the storage temperature, and the period ration at 362g/ 100 ml) and cesium chloride (70g/100 ml). of storage. These salts were substituted (in equal mass) for the am- [0067] Under more stringent incubation conditions, a monium sulfate in the solution described in Example 2 higher concentration of ammonium sulfate is necessary and processed liver samples as above. Cesium sulfate for preservation of the RNA. For example, 55g /100 ml 50 and cesium chloride offered partial protection to the liver is required for protection of RNA in tissue samples at RNA at 4°C. Taken together, the data suggest that of the 25°C and, as described in Example 6 70g/100 ml appears salts tested, ammonium sulfate has superior ability to required for maximal protection of RNA in tissue samples protect RNA from degradation in intact tissues. However, at 37°C. Further, if samples are stored in 55g or less the partial protection the inventors see with high concen- ammonium sulfate at 37 degrees for 24 hours, at least 55 trations of other salt suggests a shared mechanism of some studies yield partially degraded RNA as judged by action, with varying degrees of efficacy. Data is shown a 1:1 ratio of 28S:18S rRNA. Data is shown in FIG. 3. in FIG. 5

9 17 EP 1 657 313 B1 18

Example 8 This RNA is still a suitable substrate for Nuclease Pro- tection Assays or RT-PCR (Reverse Transcriptase- RNA isolated from mammalian tissues stored at 4°C Polymerase Chain Reaction) both of which procedures in RNAlater™ are known to those of skill in the art. Data is shown in 5 FIG. 8. [0070] Fresh mouse brain, heart, kidney, liver, and spleen samples were placed in RNA later ™ solution, pre- Example 11 pared as taught in Example 2, and stored at 4°C. After incubation of one week, 2 weeks, and 4 weeks, RNA was RNA isolated from amphibian, fish, insect, bacteria, extracted from the tissue samples and analyzed by de- 10 and plant tissue stored at 4°C in RNAlater™ naturing agarose gel electrophoresis. All the RNA samples were intact. Fresh mouse brain, kidney, liver, and [0074] The Inventor tested the effectiveness of spleen samples were placedin 10 volumesof RNA later™ RNAlater™ to preserve the RNA in Xenopus heart and and stored at 4°C. After one week, two week, and four liver, goldfish liver, whole beetle, whole Drosophila, E. week incubations, equivalent weight fragments of tissue 15 Coli, tobacco, and alfalfa. Each were placed in RNAlat- were removed and processed. Each tissue sample was er™ solution and stored at 4°C. After incubation for 24 placed in a guanidinium isothiocyanate lysis solution, ho- hours, RNA was extracted from the samples and ana- mogenized, and isolated using the Ambion RNAque- lyzed by denaturing agarose gel electrophoresis as de- ous™ kit (as described in Example 20). The concentra- scribed in Example 1. All RNAs appeared intact. This tion of the RNA was determined by measuring the ab- 20 demonstrates the effectiveness of RNA later™ as a gen- sorbence at O.D.260. The RNA was analyzed by formal- eral reagent useful for tissues from diverse organisms. dehyde-agarose gel electrophoresis as described in Ex- ample 1. Example 12 [0071] All RNA samples were judged "intact" based on clean, sharp ribosomal RNA bands in a 2: 1 ratio of 28S: 25 Demonstration of the suitability of RN Alater™ RNA 18S. In addition, overall quality was judged by a visible as target in Northern hybridization analysis background smear of heterogeneous RNA and a lack of visible breakdown products from ribosomal RNA. Data [0075] Fresh mouse liver, kidney, and spleen samples, is shown in FIG. 6 were incubated in RNAlater™, for 24 hours at 25°C or 30 37°C. RNA was extracted, resolved by denaturing aga- Example 9 rose gel electrophoresis, transferred to a positively charged nylon membrane (Brightstar Plus™, Ambion, RNA isolated from mammalian tissues stored at am- Austin, Texas), and hybridized with a radiolabeled anti- bient temperature (25°C) in RN Alater™ sense β Actin RNA probe according to manufacturers 35 recommendations (Northern Max™ Kit, Ambion, Austin, [0072] Fresh mouse brain, heart, kidney, liver, and Texas). The discrete signal corresponding to the 1.8 kilo- spleen samples were placed in RNA later™ solution and base β Actin transcript detected in all samples indicate stored at 25°C (ambient temperature). After incubation that the messenger RNA in all samples was completely for two or four weeks, RNA was extracted from the tissue intact andavailable for hybridization bynorthern analysis. samples and analyzed by denaturing agarose gel elec- 40 trophoresis as described above. The RNA recovered af- Example 13 ter two weeks was intact. RNA recovered after a one month incubation was still ~ 50% intact as judged by the The suitability of RNAlater™ RNA as template for appearance of the 18S and 28S ribosomal RNA bands. RT-PCR analysis Data is shown in FIG. 7 45 [0076] RNA prepared from mouse liver, spleen, kid- Example 10 ney, and testis stored in RNA later™ for 24 hours at 37°C was used as template for RT-PCR with two pairs of RNA isolated from mammalian tissue stored at ex- PCR™ primers for constituitively expressed genes (the treme temperatures (37°C) in RNAlater™ 50 housekeeping genes cyclophilin and RIG/S15) in a mul- tiplex RT-PCR amplification. The expected products [0073] Fresh mouse liver, kidney, and spleen samples (216bp cyclophilin, 324bp RIG/S15) were obtained in all were placed in RNAlater™ solution and stored at 37°C. cases. Thus, the recovered RNA is suitable for RT- PCR After incubation for three days, RNA was extracted from analysis. the tissue samples and analyzed by denaturing agarose 55 gel electrophoresis, as described above. The RNA isolated was intact. The RNA isolated from tissues incubated one week - 10 days is partially degraded (~50% intact).

10 19 EP 1 657 313 B1 20

Example 14 the sample will be of poor quality. Developmental biologists studying early mouse embryonic development must Use of RNA Preservation Media to Protect Clinical perform meticulous dissections to isolate early embryos Samples from the decidua of the uterus. In another example, neu- 5 roanatomists must perform complicated dissections to [0077] There is an increasing trend towards genetic remove a specific part of a brain for RNA analysis. In an analysis of human clinical samples by RT-PCR. These anticipated use, RNA later™ or other inventive RNA pres- clinical samples include solid tumors, isolated cells, se- ervation media would be a very effective dissection me- rum, urine, blood, or feces. Biopsies of solid tumors are dium. Immersing a sample in RNA later™ or other inven- frequently analyzed for the expression of specific indica- 10 tive RNA preservation media will protect the RNA during tor genes like p53, whose aberrant expression plays a dissection, while preserving sample integrity (other rea- pivotal role in a number of cancers. White blood cells gents, such as guanidinium would cause extensive cel- isolated from normal or leukemic blood are frequently lular lysis and compromise the dissection) . Using analyzed for expression of interleukin genes. Urine, RNAlater™ or other inventive RNA preservation media blood, serum, plasma, and feces are frequently analyzed 15 would facilitate a lengthy dissection of a sample without for the presence of pathogenic organisms. In an antici- the fear that RNA in the sample will be degraded. pated use, RNA later™ or other inventive RNA preservation media can be used as a holding buffer for patient Example 17 specimens isolated in a clinical setting. The RNA in these samples would then be protected until the samples could 20 Useof RNA Preservation Media to Preserve Samples be transferred to a laboratory setting where intact RNA for Pathogenic Testing would be extracted for analysis. In an anticipated use, RNAlater™ or other inventive RNA preservation media [0080] Nucleic acid analysis to detect and classify can be used to stabilize normally unstable viral RNA pathogenic organisms is a fast growing technology. In (such as HIV) in blood products for subsequent diagno- 25 an anticipated use. RNAlater™ or other inventive RNA sis. preservation media may be used as a holding buffer for specimens collected by health inspectors in the field. Example 15 Placing samples in RNAlater™ or other inventive RNA preservation media would preserve the RNA in the sam- Use of RNA Preservation Media to Preserve Field 30 ples. The samples could then be analyzed using nucleic Specimens acid technology for the presence of pathogenic organisms. For example, a USDA inspector could collect sam- [0078] Field biologists throughout the world face the ples of meat from a slaughterhouse and place them in common problem of how to preserve samples collected RNAlater™ or other inventive RNA preservation media. in the field for later analysis in the laboratory. Often re- 35 The RNA in pathogenic organisms present on the surface search into RNA expression is simply avoided due to the of the sample would be preserved intact. The meat can logistical difficulties involved in maintaining specimens later be removed from the sample, the pathogens recov- frozen in dry ice or liquid nitrogen. In an anticipated use, ered from the RNAlater™ or other inventive RNA pres- RNAlater™ or other inventive RNA preservation media ervation media, and the RNA isolated for analysis. One may be used as a holding buffer for specimens isolated 40 important feature of RNAlater™ or other inventive RNA in the field by scientists who need to preserve samples preservation media is that it is bactericidal, but does not until a later return to the laboratory environment. An im- lyse cells. Therefore, the titer of pathogenic organisms portant benefit of RNAlater™ or other inventive RNA will not change during storage of samples and skew ap- preservation media is that small specimens (such as mi- parent numbers, and samples can also be analyzed microorganisms) remain intact. Thus, complex specimens 45 croscopically for additional information. such as a population of microorganisms isolated from water samples can be preserved en masse, sorted by Example 18 classification in the laboratory, then analyzed for gene expression. Use of RNA Preservation Media to Preserve Patho- 50 gens Example 16 [0081] Thousands of specimens each year are stored Use of RNA Preservation Media as a Dissection Me- and shipped on dry ice to central laboratories that perform dium RNA analysis to detect pathogens. In an anticipated use, 55 RNAlater™ or other inventive RNA preservation media [0079] Biologists must frequently perform a complicat- may be used as a shipping buffer, preserving RNA in ed dissection in order to isolate a specimen for RNA anal- infectious, disease causing organisms. RNAlater™ or ysis. Often the time delay means that RNA isolated from other inventive RNA preservation media will permit sam-

11 21 EP 1 657 313 B1 22 ples to be shipped at ambient or near- ambient tempera- ple, any known methods of isolating RNA, such as those tures without fear of RNA degradation. An organization of Boom et al. (selective binding and retention of nucleic that would benefit both by the extra degree of protection acid to glass matrix - Qiaprep™, Qiagen, Inc.), Chomc- RNAlater™ or other inventive RNA preservation media zynski et al. (Trizol™, MRC), Macfarlane et al. (Catrimox would afford, plus realize a significant savings in shipping 5 14™, Iowa Biotechnology, Inc.), Bugos et al. (Guanidine: costs is The Centers For Disease Control (CDC), who Lithium Chloride), or Auffray et al. (LiCl:urea extraction), receive many human and animal samples for pathogenic or kits for the practice of such methods may be used in testing. this regard. [0085] The following specific examples describe the Example 19 10 use of Ambion kits to isolate RNA from tissue samples or cells stored in RNA later™ or other inventive RNA pres- Use of RNA Preservation Media in FACS Sorting ervation media. It is contemplated that Ambion may choose to sell a combined kit for the preservation of RNA [0082] Fluorescent Activated Cell Sorting (FACS) is a in tissue samples or cells, followed by the subsequent method with which cells in suspension can be separated 15 isolation of RNA from those samples. based on differences in cell surface markers. We antic- ipate the use of RNA later™ or other inventive RNA pres- Example 22 ervation media as a suspension solution for cells to be run through a FACS machine. In this way, the RNA in Isolation of Cellular RNA from Samples Preserved the cells would be preserved. Once the cells are sorted, 20 in RNAlater™ or other inventive RNA preservation intact RNA can be isolated for analysis. media Using the Ambion ToTally RNA™ Kit.

Example 20 [0086] The Ambion ToTally™ RNA kit is a Guanidin- ium/Acid Phenol method of preparing cellular RNA. Use of RNA Preservation Media to Preserve Soil Bac- 25 [0087] In order to demonstrate the utility of combining teria RNAlater™ or other inventive RNA preservation media preservation of tissue sample with the ToTally RNA™ [0083] With the advent of RT-PCR methodology to procedure, tissue samples were removed from storage identify bacterial species, there is a growing need for in RNAlater™ and homogenized in 10 volumes of a Gua- methods to isolate soil bacteria away from soil for sub- 30 nidinium Isothiocyanate lysis solution consisting of 4M sequent RNA isolation. However, RNA in bacteria is ex- guanidinium hydrochloride, 0.5% sarcosine, 25 mM So- tremely labile and is rapidly degraded during the isolation dium Citrate, 0.1M 2-mercaptoethanol. Proteins are re- protocol. An anticipated use of RNAlater™ or other in- moved by an extraction with an equal volume of phenol: ventive RNA preservation media is as a first step in RNA chloroform (1:1), followed by a centrifugation to separate isolation from soil bacteria. Soil can be dispersed in35 the aqueous and organic phases. The aqueous phase is RNAlater™ or other inventive RNA preservation media, recovered and extracted a second time with phenol at instantly protecting the RNA within bacteria, but keeping pH 4.7. This second extraction partitions any remaining the bacteria intact. The soil can then be safely removed proteins in the organic phase and the low pH forces any by low speed centrifugation, then the bacteria can be DNA into the organic phase. The RNA remains in the recovered by centrifugation at higher speed. The RNA lat- 40 aqueousphase. The aqueous phase is recovered bycen- er™ or other inventive RNA preservation media will pre- trifugation. The RNA is recovered from the aqueous vent the RNA from being degraded during the isolation phase by precipitation with 0.3M Sodium Acetate and 2.5 procedure. volumes of ethanol. Results are shown in FIG. 9.

Example 21 45 Example 23

Methods of Isolating RNA Preserved in RNAlater™ Isolation of Cellular RNA from Samples Preserved or other inventive RNA preservation media in RNAlater™ or other inventive RNA preservation media Using the Ambion RNAqueous™ Kit. [0084] Several methods of isolating RNA from tissue 50 samples have been evaluated for suitability with RNA [0088] The Ambion RNAqueous™ kit is a Guanidinium preservation media-preserved tissues. Several such lysis method for isolating RNA that does not require or- methods can be practiced with kits available from Ambi- ganic solvents. Instead, it relies upon the selective ad- on. Of course, Ambion kits are not required for the iso- sorption of RNA upon a glass fiber filter. lation of RNA from RNA preservation media-preserved 55 [0089] In order to demonstrate the utility of combining tissues, and the use of other methodologies or kits to RNAqueous ™ with RNAlater™ or other inventive RNA isolate RNA from RNA preservation media- preserved tis- preservation media tissue samples were removed from sues and cell is covered by this specification. For exam- storage in RNAlater™, transferred directly to the Gua-

12 23 EP 1 657 313 B1 24 nidinium Isothiocyanate lysis solution provided in imentation in light of the present disclosure. RNAqueous™ and homogenized. The homogenate was applied to a glass fiber filter and washed with several REFERENCES buffers, which remove protein and DNA. Pure RNA was then eluted from the filter with water. The coupling of 5 [0093] The following references, to the extent that they RNAlater™ or other inventive RNA preservation media provide exemplary procedural or other details supple- and RNAqueous™ has the advantage that no caustic or mentary to those set forth herein, are listed: carcinogenic organic reagents (phenol, chloroform, eth- U.S. Patent 5,922,591 anol, acetic acid, etc) are used throughout the procedure. U.S. Patent 5,921,396 Fresh samples or samples preserved in RNAlater™ were 10 U. S. Patent 5,726,012 homogenized in 10 volumes of a solution consisting of U.S. Patent 5,090,420 4M guanidinium hydrochloride, 1% sarcosine. 25 mM So- Allewell, N.M., Sama, A., "The effect of ammonium sul- dium Citrate, 0.1M 2- mercaptoethanol, 2% Triton X- 100. fate on the activity of ribonuclease A." Biochemica et Bi- The homogenate is diluted 2X and passed over a glass ophysica Acta 341:484-488 (1974). fiber filter. Under these conditions, nucleic acids bind to 15 Auffray, C., Rougeon, F., "Purification of mouse immu- the filter and proteins are washed away. Several sequen- noglobulin heavy chain messenger RNAs from total my- tial washes in high salt and ethanol differentially wash eloma tumor RNA." Eur. J. Biochemistry Jun;107(2) away the DNA, leaving only RNA bound to the filter. The 303-314 (1980). RNA is subsequently recovered by elution with hot water. Boom, W.R., Adriaanse, H., Kievits, T., Lens, P.F., US Results are shown in FIG. 9. 20 Patent No. 5,234,809 entitled: Process for Isolating Nu- cleic Acid. Example 24 Bugos R.C., Chiang, V.L., Zhang, X.H., Campbell. E.R., Podila G.K., Campbell W.H., "RNA isolation from plant Use of solid RNAlater to preserve nucleic acid in liq- tissues recalcitrant to extraction by guanidine." Biotech- uid samples 25 niques Nov;19(5)734-7 (1995). Cairns, M.T., Church, S., Johnston, P.G., Phenix, K.V., [0090] The solid components of the preferred RNA Marley, J.J., "Paraffin-embedded tissues as a source of preservation composition may be dissolved directly in a RNA for gene expression analysis in oral malignancy." liquid sample to be stabilized, added to a sample/liquid Oral Diseases Sep;3(3):157-161 (1997). mixture, added to a sample that has been placed in a 30 Chomczynski, P., US Patent No. 5,346,994 entitled: liquid or present in a collection vessel prior to collection Shelf Stable Product and Process for Isolating RNA, of a sample or sample/liquid mixture. DNA, and Proteins. [0091] The solid components can be blended in a dry Dimulescu et al., "Characterization of RNA in Cytologic mixer to the proper composition such that when added Samples Preserved in a Methanol- Based Collection So- to an aqueous sample and dissolved, the preferred salt, 35 lution," Molecular Diagnosis 3(2):67-72 (1998). buffer, and chelator concentration is achieved in solution. Esser, et al., "Isolation of full- sized mRNA from ethanol- The pre-measured aliquots of powdered, dry compo- fixed cells after cellular immunofluorescence staining nents can be loaded into sample collection vessels, and and fluorescence-activated cell sorting." (FACS). Cytom- an appropriate volume of sample would be added. The etry 1995 Dec 1;21(4):382-386. collection vessel would then be agitated, dissolving the 40 Foss, R.D., Guha-Thakurta, N., Conran, R.M., Gutman, RNAlater components in the solution. This would mini- P., "Effects of fixative and fixation time on the extraction mize operator exposure to the sample, and would be a and polymerase chain reaction amplification of RNA from preferred method for stabilizing nucleic acids in a biolog- paraffin-embedded tissue." Comparison of two house- ical specimen such as blood or urine. Alternatively, the keeping mRNA controls. Diagn Mol Pathol Sep;3(3): dry components can be pressed into tablet form and45 148-155 (1994). stored in bulk. Tablets would be a convenient format for Houze, T.A., Gustavsson, B., "Sonification as a means storage and can be added in the correct quantity to a of enhancing the detection of gene expression levels sample of any size in any type of container. This would from formalin-fixed, paraffin-embedded biopsies." Bio- be a preferred method for field collection of liquid samples techniques Dec;21(6):1074-1078 (1996). from such sources as water reservoirs or sewage plants 50 Hurley et al., US Paten No. 5,256,571 entitled Cell Pres- or the dairy industry. The primary advantages of using ervation Solution (1993) the RNA preservation reagent in dry mode would be Koopmans, M., Monroe S.S., Coffield, L.M., and Zaki, weight savings in storage and transport, spills of solid S.R., "Optimization of extraction and PCR amplification components would be less likely, and volume savings in of RNA extracts from paraffin- embedded tissue in differ- that preserving liquid samples with dry reagents would 55 ent fixatives." J.Virol Methods Jul; 43 (2):189-204 (1993). minimize the final volume of the sample. Lin L.-N., Brandts, J.F., "Refolding of ribonuclease in the [0092] All of the compositions and methods disclosed presence and absence of ammonium sulfate pulses." herein can be made and executed without undue exper- Comparison between experiments and simulations. Bi-

13 25 EP 1 657 313 B1 26 ochemistry 26:1826-1830 (1987) liquid is water. Macfarlane, D.E., US Patent No. 5,010,183 entitled: Process for Purifying DNA and RNA using Cationic De- 14. The method of any of claims 1 to 12, wherein the tergents. liquid is a buffer. Stanta G., Schneider, C., "RNA extracted from paraffin- 5 embedded human tissues is amendable to analysis by 15. The method of any of claims 1 to 14, wherein the PCR amplification." Biotechniques Sep; (3): 11304 RNA preservation composition has a final ammoni- (1991). um sulfate concentration of at least 35g/100ml.

10 16. The method of any of claims 1 to 15, further com- Claims prising the step of isolating the preserved RNA.

1. A method of preserving RNA in a sample comprising: 17. The method of claim 16, wherein the RNA is isolated at a temperature that is greater than -20 ° C. - obtaining an RNA-containing sample; 15 - providing a salt defined as ammonium sulfate, 18. The method of claim 16 or 17, wherein the sample and is stored prior to the isolation of the RNA. - admixing the sample, the salt, and a liquid to form an RNA preservation composition having 19. The method of claim 18, wherein the sample is stored a final ammonium sulfate concentration from 20 unfrozen at -20°C to 45°C. 30g/100ml to 40g/100ml. 20. The method of claim 18, wherein the sample is stored 2. The method of claim 1, wherein the sample is com- at greater than 0°C. prised in a liquid prior to admixing the sample with the ammonium sulfate. 25 21. Use of an RNA preservation medium comprising a salt constituted by ammonium sulfate at a concen- 3. The method of claim 1 or 2, wherein the salt is in a tration of from 30 to 40g/100ml for preserving RNA solid form prior to admixing with the sample and the in a sample. liquid. 30 4. The method of claim 1 or 2, wherein the salt is com- Patentansprüche prised in a liquid prior to admixing with the sample. 1. Ein Verfahren zur Konservierung von RNA in einer 5. The method of any of claims 1 to 4, wherein the sam- Probe, das folgendes umfasst: ple is a blood cell and the liquid is blood serum. 35 - Gewinnen einer RNA-haltigen Probe; 6. The method of any of claims 1 to 4, wherein the sam- - Bereitstellen eines Salzes, das als Ammoni- ple is urine. umsulfat definiert ist; und - Vermischen der Probe, des Salzes und einer 7. The method of any or claims 1 to 5, wherein the sam- 40 Flüssigkeit zur Bildung einer RNA-Konservie- ple is a suspension of cells. rungszusammensetzung mit einer Endkonzen- tration an Ammoniumsulfat von 30 g/ 100 ml bis 8. The method of any of claims 1 to 4, wherein the sam- 40 g/100 ml. ple is a solid tissue sample. 45 2. Das Verfahren nach Anspruch 1, wobei die Probe 9. The method of any of claims 1 to 4, wherein the sam- vor dem Vermischen der Probe mit dem Ammoni- ple is a blood sample. umsulfat in einer Flüssigkeit umfasst ist.

10. The method any of claims 1 to 4, wherein the sample 3. Das Verfahren nach Anspruch 1 oder 2, wobei das is a water sample. 50 Salz vor dem Vermischen mit der Probe und der Flüssigkeit in einer festen Form vorliegt. 11. The method of any of claims 1 to 4, wherein the sample comprises an entire organism. 4. Das Verfahren nach Anspruch 1 oder 2, wobei das Salz vor dem Vermischen mit der Probe in einer Flüs- 12. The method of claim 11, wherein the organism is a 55 sigkeit umfasst ist. pathogen within a tissue sample or other organism. 5. Das Verfahren nach einem der Ansprüche 1 bis 4, 13. The method of any of claims 1 to 12, wherein the wobei die Probe eine Blutzelle und die Flüssigkeit

14 27 EP 1 657 313 B1 28

Blutserum ist. Revendications

6. Das Verfahren nach einem der Ansprüche 1 bis 4, 1. Procédé de conservation de l’ARN dans un échan- wobei die Probe Urin ist. tillon, comprenant : 5 7. Das Verfahren nach einem der Ansprüche 1 bis 5, - l’obtention d’un échantillon contenant de wobei die Probe eine Zellsuspension ist. l’ARN ; - la fourniture d’un sel défini comme un sulfate 8. Das Verfahren nach einem der Ansprüche 1 bis 4, d’ ammonium ; et wobei die Probe eine feste Gewebeprobe ist. 10 - le mélange de l’échantillon, du sel et d’un liquide pour former une composition de conservation 9. Das Verfahren nach einem der Ansprüche 1 bis 4, de l’ARN ayant une concentration finale en sul- wobei die Probe eine Blutprobe ist. fate d’ammonium de 30g/100ml à 40g/100ml.

10. Das Verfahren nach einem der Ansprüche 1 bis 4, 15 2. Procédé selon la revendication 1, dans lequel wobei die Probe eine Wasserprobe ist. l’échantillon est compris dans un liquide avant le mé- lange de l’échantillon avec le sulfate d’ammonium. 11. Das Verfahren nach einem der Ansprüche 1 bis 4, wobei die Probe einen gesamten Organismus um- 3. Procédé selon la revendication 1 ou 2, dans lequel fasst. 20 le sel est sous une forme solide avant le mélange avec l’échantillon et le liquide. 12. Das Verfahren nach Anspruch 11, wobei der Orga- nismus ein Pathogen in einer Gewebeprobe oder 4. Procédé selon la revendication 1 ou 2, dans lequel einem anderen Organismus ist. le sel est compris dans un liquide avant le mélange 25 avec l’échantillon. 13. Das Verfahren nach einem der Ansprüche 1 bis 12, wobei die Flüssigkeit Wasser ist. 5. Procédé selon l’une quelconque des revendications 1 à 4, dans lequel l’échantillon est une cellule san- 14. Das Verfahren nach einem der Ansprüche 1 bis 12, guine et le liquide est un sérum sanguin. wobei die Flüssigkeit ein Puffer ist. 30 6. Procédé selon l’une quelconque des revendications 15. Das Verfahren nach einem der Ansprüche 1 bis 14, 1 à 4, dans lequel l’échantillon est de l’urine. wobei die RNA-Konservierungszusammensetzung eine Endkonzentration an Ammoniumsulfat von min- 7. Procédé selon l’une quelconque des revendications destens 35 g/100 ml aufweist. 35 1 à 5, dans lequel l’échantillon est une suspension de cellules. 16. Das Verfahren nach einem der Ansprüche 1 bis 15, das ferner den Schritt der Isolierung der konservier- 8. Procédé selon l’une quelconque des revendications ten RNA umfasst. 1 à 4, dans lequel l’échantillon est un échantillon de 40 tissu solide. 17. Das Verfahren nach Anspruch 16, wobei die RNA bei einer Temperatur isoliert wird, die größer als -20 9. Procédé selon l’une quelconque des revendications °C beträgt. 1 à 4, dans lequel l’échantillon est un échantillon de sang. 18. Das Verfahren nach Anspruch 16 oder 17, wobei die 45 Probe vor der Isolierung der RNA gelagert wird. 10. Procédé selon l’une quelconque des revendications 1 à 4, dans lequel l’échantillon est un échantillon à 19. Das Verfahren nach Anspruch 18, wobei die Probe base d’eau. ungefroren bei -20 °C bis 45 °C gelagert wird. 50 11. Procédé selon l’une quelconque des revendications 20. Das Verfahren nach Anspruch 18, wobei die Probe 1 à 4, dans lequel l’échantillon comprend un orga- bei mehr als 0 °C gelagert wird. nisme entier.

21. Verwendung eines RNA-Konservierungsmediums 12. Procédé selon la revendication 11, dans lequel l’or- umfassend ein Salz, das von Ammoniumsulfat ge- 55 ganisme est un pathogène dans un échantillon de bildet wird, bei einer Konzentration von 30 bis 40 g/ tissu ou un autre organisme. 100 ml zur Konservierung von RNA in einer Probe. 13. Procédé selon l’une quelconque des revendications

15 29 EP 1 657 313 B1 30

1 à 12, dans lequel le liquide est de l’eau.

14. Procédé selon l’une quelconque des revendications 1 à 12, dans lequel le liquide est un tampon. 5 15. Procédé selon l’une quelconque des revendications 1 à 14, dans lequel la composition de conservation de l’ARN a une concentration finale en sulfate d’ammonium d’au moins 35g/100ml. 10 16. Procédé selon l’une quelconque des revendications 1 à 15, comprenant en outre l’étape d’isolement de l’ARN conservé.

17. Procédé selon la revendication 16, dans lequel15 l’ARN est isolé à une température qui est supérieure à -20°C.

18. Procédé selon la revendication 16 ou 17, dans lequel l’échantillon est stocké avant l’isolement de l’ARN. 20

19. Procédé selon la revendication 18, dans lequel l’échantillon est stocké non congelé à -20°C à 45°C.

20. Procédé selon la revendication 18, dans lequel25 l’échantillon est stocké à plus de 0°C.

21. Utilisation d’un milieu de conservation de l’ARN comprenant un sel constitué par le sulfate d’ammonium à une concentration de 30 à 40g/ 100ml pour conser- 30 ver l’ARN dans un échantillon.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

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Non-patent literature cited in the description

• Allewell, N.M. ; Sama, A. The effect of ammonium • Foss, R.D. ; Guha-Thakurta, N. ; Conran, R.M. ; sulfate on the activity of ribonuclease A. Biochemica Gutman, P. Effects of fixative and fixation time on et Biophysica Acta, 1974, vol. 341, 484-488 [0093] the extraction and polymerase chain reaction ampli- • Auffray, C. ; Rougeon, F. Purification of mouse im- fication of RNA from paraffin-embedded tissue. Com- munoglobulin heavy chain messenger RNAs from to- parison of two housekeeping mRNA controls. Diagn tal myeloma tumor RNA. Eur. J. Biochemistry Jun, Mol Pathol Sep, 1994, vol. 3 (3), 148-155 [0093] 1980, vol. 107 (2), 303-314 [0093] • Houze, T.A.; Gustavsson, B. Sonification as a • Bugos R.C. ; Chiang, V.L. ; Zhang, X.H. ; Camp- means of enhancing the detection of gene expression bell. E.R. ; Podila G.K. ; Campbell W.H. RNA iso- levels from formalin-fixed, paraffin-embedded biop- lation from plant tissues recalcitrant to extraction by sies. Biotechniques Dec, 1996, vol. 21 (6), guanidine. Biotechniques Nov, 1995, vol. 19 (5), 1074-1078 [0093] 734-7 [0093] • Koopmans, M. ; Monroe S.S. ; Coffield, L.M. ; Za- • Cairns, M.T. ; Church, S. ; Johnston, P.G. ; henix, ki, S.R. Optimization of extraction and PCR amplifi- K.V. ; Marley, J.J. Paraffin-embedded tissues as a cation of RNA extracts from paraffin-embedded tis- source of RNA for gene expression analysis in oral sue in different fixatives. J.Virol Methods Jul, 1993, malignancy. Oral Diseases Sep, 1997, vol. 3 (3), vol. 43 (2), 189-204 [0093] 157-161 [0093] • Lin L.-N. ; Brandts, J.F. Refolding of ribonuclease • Dimulescu et al. Characterization of RNA in Cyto- in the presence and absence of ammonium sulfate logic Samples Preserved in a Methanol-Based Col- pulses. Comparison between experiments and sim- lection Solution. Molecular Diagnosis, 1998, vol. 3 ulations. Biochemistry, 1987, vol. 26, 1826-1830 (2), 67-72 [0093] [0093] • Esser et al. Isolation of full-sized mRNA from etha- • Stanta G. ; Schneider, C. RNA extracted from par- nol-fixed cells after cellular immunofluorescence affin-embedded human tissues is amendable to anal- staining and fluorescence-activated cell sorting. ysis by PCR amplification. Biotechniques Sep, 1991, (FACS). Cytometry, 01 December 1995, vol. 21 (4), vol. 11 (3), 304 [0093] 382-386 [0093]