Methods and compositions for preparing tissue samples for RNA extraction
View Patent ↗The present invention concerns the methods and compositions for preparing a tissue section or biological sample, particularly to preserve RNA in the section or sample, by not exposing or contacting the sample or section to a solution that is composed of mostly water. Tissue sections can be fixed, stained, and dehydrated for subsequent manipulation, including laser capture microdissection (LCM) for further analysis using methods and/or compositions of the invention.
1. A method for extracting nucleic acid molecules from a tissue section comprising:
a) contacting a tissue section with a first set of one or more solutions comprising no more than about 50% water;
b) contacting the tissue section with one or more stain in a solution comprising no more than about 50% water;
c) contacting the tissue section with a second set of one or more solutions comprising no more than about 50% water; and
d) then extracting nucleic acid molecules from the tissue section.
2. The method of claim 1 , wherein the stain is in a solution with less than about 10% water.
3. The method of claim 1 , wherein one or more of the solutions is at least about 70% alcohol.
4. The method of claim 3 , wherein one or more of the solutions is at least about 90% alcohol.
5. The method of claim 4 , wherein one or more of the solutions is about 100% alcohol.
6. The method of claim 3 , wherein the stain is in a solution that is at least about 80% alcohol.
7. The method of claim 6 , wherein the stain is in a solution that is about 100% alcohol.
8. The method of claim 3 , wherein the alcohol in at least one of the solutions comprises an alcohol selected from the group consisting of ethanol, methanol, and propanol.
9. The method of claim 1 , wherein solutions comprising water include water that has been treated with diethyl pyrocarbonate (DEPC).
10. The method of claim 1 , wherein at least one of the solutions in the second set comprises a non-alcohol organic solvent.
11. The method of claim 10 , wherein the organic solvent solution is at least about 50% organic solvent.
12. The method of claim 11 , wherein the organic solvent solution is at least about 75% organic solvent.
13. The method of claim 12 , wherein the organic solvent solution is at least about 90% organic solvent.
14. The method of claim 13 , wherein the organic solvent solution is about 100% organic solvent.
15. The method of claim 10 , wherein the organic solvent is a hydrocarbon selected from the group consisting of xylene or toluene.
16. The method of claim 1 , wherein the stain is selected from the group consisting of cresyl violet acetate, hematoxylin, and eosin.
17. The method of claim 1 , wherein the tissue sample is exposed to more than one stain.
18. The method of claim 1 , further comprising performing laser capture microscopy on the tissue section before step (a) or before step (d).
19. The method of claim 18 , further comprising extracting nucleic acid molecules from cells of the tissue section that have been subjected to laser capture microdissection.
20. The method of claim 19 , wherein the nucleic acid molecules include ribonucleic acids (RNA).
21. The method of claim 19 , wherein the nucleic acid molecules include RNA.