Methods for fixing and detecting RNA
The invention provides a method for fixing an RNA molecule in a biological sample. The method comprises contacting the biological sample with an aldehyde-containing fixative, and subsequently contacting the sample with a solution comprising a carbodiimide and a heterocyclic derivative selected from the group consisting of an imidazole, pyrazole, triazole or tetrazole or a combination thereof. A method for differentiating cells in a biological sample is also provided. A method for quantification of RNA retention in a biological sample is also provided. A kit for fixing RNA in a sample is provided as well.
1. A method for fixing an RNA molecule in a biological sample, said method comprising contacting the biological sample with an aldehyde-containing fixative, and subsequently contacting the sample with an aqueous solution comprising a carbodiimide and a heterocyclic derivative selected from the group consisting of an pyrazole, triazole or tetrazole or a combination thereof.
2. The method of claim 1 , wherein said heterocyclic derivative has at least one of the following structures:
wherein R 1 , R 2 , and R 3 independently are H, alkyl, aryl, F, CI, Br, I, CN, NO 2 , NH 2 , OH, SH, ethylthio-, alkyloxy-, or carbonyl.
3. The method of claim 1 , wherein the aldehyde-containing fixative is formaldehyde, a derivative of formaldehyde, paraformaldehyde, glyoxal, glutaraldehyde, or combination thereof.
4. The method of claim 1 , wherein the heterocyclic derivative is selected from the group consisting of 1-hydroxyl-benzotriazole and 5-ethylthiotetrazole.
5. The method of claim 4 , wherein the heterocyclic derivative is 5-ethylthiotetrazole.
6. The method of claim 1 , wherein the carbodiimide is selected from the group consisting of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl), 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate (CMC), N,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide methiodide (EDC-Mel).
7. The method of claim 6 , wherein the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide methiodide (EDC-Mel).
8. The method of claim 1 , wherein the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide methiodide (EDC-Mel) and the heterocyclic derivative is 5-ethylthiotetrazole.
9. The method of claim 8 , wherein the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide methiodide (EDC-Mel) has a concentration of about 20 mM to about 300 mM.
10. The method of claim 9 , wherein the solution comprising 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide methiodide (EDC-MeI) contacts the sample at a temperature of about 20° C. to about 70° C.
11. The method of claim 1 , wherein the solution comprising a carbodiimide and a heterocyclic derivative and has a pH of about 6.0 to about 10.0.
12. The method of claim 1 , wherein the solution comprising a carbodiimide and a heterocyclic derivative has a pH of about 7.0 to about 8.0.
13. The method of claim 1 , wherein the RNA molecule is a short RNA.
14. The method of claim 1 , wherein the RNA molecule is a miRNA.
15. The method of claim 1 , wherein the solution comprising a carbodiimide and a heterocyclic derivative further comprises a detergent.
16. The method of claim 15 , wherein the detergent is selected from the group consisting TWEEN20 (Polysorbate20), TWEEN80 (Polysorbate80), TRITON A-100 (Octylphenol ethoxylate), TRITON-114 (ter-octylphenoxypoly(ethoxyethanol)), Digitonin, Saponin, CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate), Denhardt's solution, Heparin, BRIJ35 (Polyethylene glycol hexadecylether), Sodium dodecyl sulfate (SDS), and urea.
17. The method of claim 1 , further comprising contacting the solution comprising a carbodiimide and a heterocyclic derivative with a cyanogen halide.
18. The method of claim 17 , wherein the cyanogen halide is selected from the group consisting of cyanogen bromide, cyanogen iodide, cyanogen chloride and cyanogen fluoride.
19. The method of claim 17 , wherein the cyanogen halide is cyanogen bromide or cyanogen iodide.
20. A method for detecting a target RNA molecule in a biological sample, said method comprising
a. contacting the biological sample with an aldehyde-containing fixative;
b. subsequently contacting the sample with a solution comprising a carbodiimide and a heterocyclic derivative to produce crosslinked RNA in the sample, said heterocyclic derivative selected from the group consisting of an pyrazole, triazole or tetrazole or a combination thereof;
c. contacting the sample with a probe, said probe being complementary to all or a part of a region of interest of the RNA in the sample, thereby producing hybridized RNA; and
d. detecting the hybridized RNA as the target RNA.
21. The method according to claim 1 , wherein the heterocyclic derivative is a tetrazol.
22. The method according to claim 20 , wherein the heterocyclic derivative is a tetrazol.