Methods and Reagents for Preserving RNA in Cell and Tissue Samples
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(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 Printed by Jouve, 75001 PARIS (FR) 1 EP 1 657 313 B1 2 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 speci- mens 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. The tissue 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.