Methods and reagents for preserving RNA in cell and tissue samples

• US 6,204,375 B1
• Filed: 07/31/1998
• Issued: 03/20/2001
• Est. Priority Date: 07/31/1998
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Ambion Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Lader, Eric S.
• Primary Examiner(s): Kunz, Gary L.
• Application Number:
  US09/127,435
• Time in Patent Office: 963 Days
• Field of Search: 435/91.3, 536/25.4, 536/25.41, 536/25.42
• US Class Current: 536/25.4
• CPC Class Codes: C12N 1/04 Preserving or maintaining viable microorganisms immobilised microorganisms C12N11/00
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence

Abstract

This specification relates to the field of molecular biology and provides a novel method and reagent for preserving and protecting the ribonucleic acid (RNA) content of tissue samples from degradation prior to RNA isolation. This preservation may be accomplished without ultra-low temperature storage or disruption of the tissue.

Claims

1. A method of preserving RNA in an intact cell comprising:

obtaining an intact cell; and

treating the cell with an RNA preservation medium comprising at least one salt, which RNA preservation medium has a total salt concentration of between 10 g/100 ml and the saturating concentration of the salt, and which RNA preservation medium infiltrates the cell and protects RNA in the cell from nucleases;

whereby said RNA is said cell is preserved at temperatures greater than −

20 degree Centigrade.

2. The method of claim 1, wherein the RNA preservation medium precipitates the RNA in the cell along with cellular protein in the cell and renders the RNA inaccessible to nucleases.

3. The method of claim 1, wherein the salt is a sulfate salt.

4. The method of claim 3, wherein the salt is ammonium sulfate.

5. The method of claim 1, wherein the salt is present in a concentration between 20 g/100 ml and the saturating concentration of the salt.

6. The method of claim 1, wherein the salt is ammonium sulfate in a concentration of between 30 g/100 ml and 80 g/100 ml.

7. The method of claim 1, wherein the RNA preservation medium comprises a combination of at least two salts.

8. The method of claim 7, wherein the total salt concentration is between 20 g/100 ml and 100 g/100 ml.

9. The method of claim 1, wherein the RNA preservation medium comprises a chelator of divalent cations.

10. The method of claim 1, wherein the RNA preservation medium comprises a buffer.

11. The method of claim 1, wherein the RNA preservation medium has a pH of between 4 and 8.

12. The method of claim 1, wherein the cell is in a suspension of cells.

13. The method of claim 1, wherein the cell is a solid tissue sample.

14. The method of claim 1, wherein the cell is in an entire organism.

15. The method of claim 14, wherein the organism is a pathogen within a tissue sample or other organism.

16. The method of claim 1, further comprising the step of isolating the preserved RNA.

17. The method of claim 16, wherein the RNA is isolated at a temperature that is greater than −

20°

C.

18. The method of claim 16, wherein the cell is stored prior to the isolation of the RNA.

19. The method of claim 18, wherein the cell is stored unfrozen at −

20°

C. to 45°

C.

20. The method of claim 19, wherein the cell is stored at greater than 0°

C.

21. The method of claim 1, wherein the RNA preservation medium comprises a combination of at least two salts.

22. The method of claim 1, wherein the RNA preservation medium further comprises an organic solvent.

23. The method of claim 15, wherein said pathogen is a virus.

24. The method of claim 1, wherein the RNA preservation medium comprises a combination of at least two salts, which salts are present in a total salt concentration of between 20 g/100 ml and 100 g/100 ml.