Stabilized RNA solutions

Patent metadata

Field	Value
Publication number	US-9896682-B2
Application number	US-201615063139-A
Country	US
Kind code	B2
Filing date	Mar 7, 2016
Priority date	Mar 6, 2015
Publication date	Feb 20, 2018
Grant date	Feb 20, 2018

Abstract

Methods and compositions are described herein for protecting RNA from autocatalytic and divalent cation induced degradation in an aqueous solution.

First claim

What is claimed is:

1. A method of protecting RNA from degradation in a solution, said method comprising:
providing a solution comprising:
i. water;
ii. RNA, wherein the RNA is purified away from endogenous host proteins of a cell or tissue from which the RNA is derived;
iii. divalent cations;
iv. a DNase enzyme, wherein the DNase enzyme is an active DNase enzyme that is exogenous to the cell or tissue from which the RNA is purified; and
v. a phosphorous-containing compound selected from the group consisting of phosphate, glycerol phosphate, glucose phosphate, ribose phosphate, and pyrophosphate;
incubating the solution at a temperature of at 40° C., wherein the incubating the solution at the temperature of at least 40° C. comprises inactivating the DNase enzyme,
wherein the RNA is protected from degradation as compared to an RNA in an aqueous solution that contains the divalent cations but does not contain the phosphorous-containing compound.

2. The method of claim 1 , wherein the providing comprises purifying RNA from the cell or tissue.

3. The method of claim 1 , wherein the DNase enzyme is at a concentration of from 0.01 U/μL to 0.2 U/μL.

4. The method of claim 1 , wherein the DNase enzyme is a recombinant DNase enzyme.

5. The method of claim 1 , wherein before the incubating the solution at a temperature of at least 40° C., the solution comprises DNA, wherein the DNA is derived from a cell or tissue and the RNA is derived from the same cell or tissue, and the method comprises incubating the solution under conditions suitable for performing a DNase reaction, thereby degrading the DNA.

6. The method of claim 5 , wherein the DNA in the solution before the DNase reaction is performed is at a concentration of less than 50 ng/μL.

7. The method of claim 1 , wherein after incubating the solution at a temperature of at least 40° C., the method comprises quantifying the RNA in the solution.

8. The method of claim 1 , wherein the phosphorous-containing compound is at a concentration of 5, mM, 10 mM, or 15 mM.

9. The method of claim 1 , wherein the solution comprises magnesium cations and calcium cations.

10. The method of claim 9 , wherein the calcium is at a concentration of from 10 μM to 1 mM and the magnesium is at a concentration of from 0.1 mM to 10 mM.

11. An aqueous solution comprising:
i. water;
ii. RNA, wherein the RNA is purified away from endogenous host proteins of a cell or tissue from which the RNA is derived;
iii. divalent cations;
iv. a DNase enzyme, wherein the DNase enzyme is an active DNase enzyme that is exogenous to the cell or tissue from which the RNA is purified; and
v. a phosphorous-containing compound selected from the group consisting of phosphate, glycerol phosphate, glucose phosphate, ribose phosphate, and pyrophosphate
wherein the RNA is protected from degradation as compared to an RNA in an aqueous solution that contains the divalent cations but does not contain the phosphorous-containing compound.

12. The solution of claim 11 , wherein the RNA is at a concentration of from 0.1 pg/mL to 500 μg/mL.

13. The solution of claim 11 , wherein the DNase enzyme is at a concentration of from 0.01 U/μL to 0.2 U/μL.

14. The solution of claim 11 , wherein the DNase enzyme is a recombinant DNase enzyme.

15. The solution of claim 11 , wherein the solution further comprises DNA.

16. The solution of claim 15 , wherein the DNA is DNase digested.

17. The solution of claim 15 , wherein the DNA in the solution is at a concentration of less than 50 ng/μL.

18. The solution of claim 15 , wherein the DNA is genomic DNA and is derived from a cell or tissue and the RNA is derived from the same cell or tissue.

19. The solution of claim 11 , wherein the solution is at a temperature of at least 40° C.

20. The solution of claim 11 , wherein the phosphorous-containing compound is at a concentration of 5 mM, 10 mM, or 15 mM.

21. The solution of claim 11 , wherein the solution comprises magnesium cations and calcium cations.

22. The solution of claim 21 , wherein the calcium is at a concentration of from 10 μM to 1 mM and the magnesium is at a concentration of from 0.1 mM to 10 mM.

23. The solution of claim 11 , wherein the solution is RNase free.

Assignees

• Bio Rad Laboratories

Inventors

• Gong Xiao Song
• Wan Cindy

Classifications

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title

• C12N15/1003 Primary

  Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title