Compositions and methods for preserving RNA in biological samples
View Patent ↗The present invention concerns methods and compositions to prepare biological samples to preserve the macromolecules in the samples. Embodiments of the invention concern the use of a soak solution that contains one or more water-miscible solvents. A sample is incubated with the soak solution to the point of saturation at a temperature above the melting temperature of the water-miscible solvent but below 0° C. The use of methods and compositions of the invention allow for subsequent preparation or analysis of the samples.
1. A method for preparing a frozen biological tissue sample for extraction of a macromolecule comprising:
saturating the frozen biological sample in a soak solution that comprises up to 20% DMSO and that is at least 60% comprised of one or more water-miscible solvents having a melting temperature below about 0° C., for a time to allow the sample to transition to a non-brittle state, wherein the saturating occurs at a temperature below 0° C., but above the melting temperature of any water-miscible solvent; and
extracting the macromolecule from the biological sample.
2. The method of claim 1 , further comprising homogenizing the sample with a homogenization solution comprising a protein denaturant after saturating the sample.
3. The method of claim 1 , wherein the soak solution is at least 70% comprised of one or more water-miscible solvents.
4. The method of claim 1 , wherein the soak solution is at least 80% comprised of one or more water-miscible solvents.
5. The method of claim 1 , wherein the one or more water-miscible solvents have a melting temperature below −20° C.
6. The method of claim 5 , wherein the one or more water-miscible solvents have a melting temperature below −50° C.
7. The method of claim 6 , wherein the one or more water-miscible solvents have a melting temperature below −70° C.
8. The method of claim 7 , wherein the one or more water-miscible solvents have a melting temperature below −100° C.
9. The method of claim 1 , wherein the soak solution contains at least two water-miscible solvents.
10. The method of claim 9 , wherein the soak solution contains at least three water-miscible solvents.
11. The method of claim 10 , wherein the soak solution contains at least four water-miscible solvents.
12. The method of claim 1 , wherein at least one of the water-miscible solvents is an alcohol.
13. The method of claim 12 , wherein the alcohol is methanol, ethanol, isopropanol, methoxyethanol, or 1-propanol.
14. The method of claim 1 , wherein at least one of the water-miscible solvents is ethyl acetate.
15. The method of claim 1 , wherein the biological sample is permeating in the soak solution up to 12 months or up to 60 months.
16. The method of claim 15 , wherein the biological sample is saturating in the soak solution up to 3 months.
17. The method of claim 16 , wherein the biological sample is saturating in the soak solution up to 1 week.
18. The method of claim 17 , wherein the biological sample is saturating in the soak solution up to 48 hours.
19. The method of claim 18 , wherein the biological sample is saturating in the soak solution for 6 to 36 hours.
20. The method of claim 19 , wherein the biological sample is saturating in the soak solution for 16 to 24 hours.
21. The method of claim 1 , wherein the soak solution further comprises a protein modification agent, or an RNase inhibitor.
22. The method of claim 21 , wherein the soak solution further comprises an RNase inhibitor.
23. The method of claim 1 , wherein the soak solution is comprised of up to 5% DMSO.
24. The method of claim 21 , wherein the soak solution further comprises a protein modification agent.
25. The method of claim 24 , wherein the soak solution comprises a protein modification agent and an RNase inhibitor.
26. The method of claim 24 , wherein the protein modification agent is an acetylating agent or a halogenating agent.
27. The method of claim 24 , wherein the soak solution is comprised of up to 5% of the protein modification agent.
28. The method of claim 21 , wherein the soak solution further comprises an RNase inhibitor.
29. The method of claim 24 , wherein saturation of the biological sample occurs at a pH in the range of about 2 to about 10.
30. The method of claim 24 , wherein saturation of the biological sample occurs at a pH in the range of about 4 to about 8.
31. The method of claim 24 , wherein saturation of the biological sample occurs at a pH in the range of about 5 to about 6.
32. The method of claim 1 , wherein nucleic acids arc extracted from the sample.
33. The method of claim 32 , wherein DNA is extracted from the sample.
34. The method of claim 32 , wherein RNA is extracted from the sample.
35. The method of claim 1 , wherein proteins are extracted from the sample.
36. The method of claim 1 , wherein carbohydrates are extracted from the sample.
37. The method of claim 1 wherein lipids are extracted from the sample.
38. The method of claim 1 , wherein a combination of macromolecules is extracted from the sample.
39. A method for extracting RNA from a frozen biological tissue sample comprising:
saturating the frozen biological sample in a soak solution that comprises up to 20% DMSO and that is at least 60% comprised of one or more water-miscible solvents having a melting temperature below about 0° C., for a time to allow the sample to transition to a non-brittle state;
homogenizing the sample with a homogenization solution comprising a protein denaturant; and,
extracting RNA from the sample after the sample is homogenized.