Methods to fix and detect nucleic acids
In one aspect, the invention relates to a method for fixing a short nucleic acid in a biological sample. In another aspect, the invention relates to a method for detecting a target short nucleic acid in a biological sample. The method includes contacting the biological sample with an aldehyde-containing fixative, and subsequently contacting the sample with a water-soluble carbodiimide. In a further aspect, the invention relates to a kit for fixing a short nucleic acid in a biological sample. The kit includes a support substrate for holding the sample; an aldehyde-containing fixative; and a water-soluble carbodiimide.
1. A method for fixing a nucleic acid in a biological sample, said method comprising
a) contacting the biological sample with an aldehyde-containing fixative; and
b) subsequently contacting the sample with a water-soluble carbodiimide, wherein said carbodiimide is in solution with a buffer.
2. The method of claim 1 , wherein the aldehyde-containing fixative is formaldehyde.
3. The method of claim 1 , wherein the carbodiimide is selected from the group consisting of: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC); 1-Cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate (CMC), N,N′-dicyclohexylcarbodiimide (DCC), and N,N′-diisopropylcarbodiimide (DIC).
4. The method of claim 3 , wherein the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.
5. The method of claim 4 , wherein the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide has a concentration of about 50 mM to about 250 mM.
6. The method of claim 4 , wherein the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide contacts the sample at a temperature of about 20° C. to about 70° C.
7. The method of claim 4 , wherein the carbodiimide has a pH of 8.0 in solution.
8. The method according to claim 1 , wherein said nucleic acid is between 5-100 nucleotides.
9. A method for detecting a target nucleic acid in a biological sample, said method comprising
a) contacting the biological sample with an aldehyde-containing fixative;
b) subsequently contacting the sample with a water-soluble carbodiimide to produce a crosslinked nucleic acid;
c) contacting the cross-linked nucleic acid with a probe, said probe being complementary to all or a part of a region of interest of the nucleic acid, thereby producing a hybridized nucleic acid; and
d) detecting the hybridized nucleic acid as the target nucleic acid.
10. The method of claim 9 , wherein the carbodiimide is selected from the group consisting of: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC); 1-Cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate (CMC), N,N′-dicyclohexylcarbodiimide (DCC), and N,N′-diisopropylcarbodiimide (DIC).
11. The method according to claim 10 , wherein the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.
12. The method according to claim 11 , wherein the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide has a concentration of about 50 mM to about 250 mM.
13. The method according to claim 11 , wherein the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide has a temperature of about 20° C. to about 70° C.
14. The method according to claim 9 , wherein the probe comprises a locked nucleic acid.
15. The method according to claim 9 wherein said nucleic acid is between 5-100 nucleotides.