Sample preparation for in situ nucleic acid analysis, methods and compositions therefor
Sample preparation processes for in situ RNA or DNA analysis, methods and compositions therefor are provided. Processes provided herein allow DNA or RNA analysis to be carried out in the same tube or on an aliquot of the prepared sample without centrifugation or extraction. The preparation process can be carried out at room temperature in as little as seven minutes and is amenable to high throughput processing using manual or robotic platforms.
1. A process for preparing a sample containing nucleic acid for in situ analysis of nucleic acid or a surrogate thereof, the process comprising:
contacting the sample containing nucleic acid with a lysis mixture under conditions and for a time to produce a lysate,
admixing the lysate with a stop mixture at substantially the same temperature as the contacting step to form a stopped mixture compatible with nucleic acid polymerase reaction conditions, and
contacting the stopped mixture with reagents for nucleic acid polymerization to form a first amplification reaction.
2. The process of claim 1 wherein the sample contains RNA and the nucleic acid polymerase is a reverse transcriptase.
3. The process of claim 1 wherein the sample contains DNA and the nucleic acid polymerase is a DNA polymerase.
4. The process of claim 1 wherein contacting the sample with a lysis mixture is for a time of 2 minutes to 30 minutes.
5. The process of claim 1 wherein the sample comprises one cell to 10 5 or 10 6 cells.
6. The process of claim 1 wherein the sample is a fixed sample.
7. The process of claim 1 wherein the sample is obtained by laser capture microdissection.
8. The process of claim 1 wherein the temperature is from 15° C. to 30° C.
9. A process for preparing a sample containing nucleic acid for in situ analysis of nucleic acid or a surrogate thereof, the process comprising:
contacting the sample containing nucleic acid with a lysis mixture under conditions and for a time to produce a lysate wherein the lysis mixture comprises a polypeptide having protease activity, and a surfactant,
admixing the lysate with a stop mixture at substantially the same temperature as the contacting step to form a stopped mixture wherein the stop mixture comprises an inhibitor of the polypeptide, and
contacting the stopped mixture with reagents for nucleic acid polymerization to form a first amplification reaction.
10. The process of claim 9 wherein the sample contains RNA and a reagent for nucleic acid polymerization is a reverse transcriptase.
11. The process of claim 9 wherein the sample contains DNA and a reagent for nucleic acid polymerization is a DNA polymerase.
12. The process of claim 9 wherein contacting the sample with a lysis mixture is for a time of 2 minutes to 30 minutes.
13. The process of claim 9 wherein the sample comprises one cell to 10 5 or 10 6 cells.
14. The process of claim 9 wherein the sample is a fixed sample.
15. The process of claim 9 wherein the sample is obtained by laser capture microdissection.
16. The process of claim 9 wherein the temperature is from 15° C. to 30° C.
17. The process of claim 9 further comprising contacting the first amplification reaction with reagents for polymerase chain reaction to generate an amplified product.
18. A process for preparing a sample containing RNA for in situ analysis of RNA or a surrogate thereof, the process comprising:
contacting the sample containing RNA with a lysis mixture under conditions and for a time to produce a lysate
wherein the lysis mixture comprises a polypeptide having deoxyribonuclease activity and a surfactant that substantially lacks fluorescence between 300 nm and 750 nm when in use for in situ analysis of RNA or a surrogate thereof, and
wherein the lysis mixture is substantially free of a cation chelator, wherein the cation chelator is EDTA.
19. The process of claim 18 wherein the lysis mixture further comprises a ribonuclease inhibitor.
20. The process of claim 18 further comprising contacting the lysate, or a portion thereof, with reagents for reverse transcription for a time and under conditions to form cDNA.
21. The process of claim 20 further comprising contacting the cDNA with reagents for PCR.
22. The process of claim 18 wherein the conditions comprise a temperature of 15° C. to 40° C.
23. The process of claim 18 wherein the time is for 3 minutes to 15 minutes.
24. The process of claim 18 wherein the sample comprises up to 10 6 cultured cells.
25. The process of claim 18 wherein the surfactant comprises octylphenol ethoxylate having an average of 7.5 ethoxylate groups.
26. The process of claim 18 wherein the surfactant comprises octylphenol ethoxylate having an average of 9.5 ethoxylate groups.
27. The process of claim 18 wherein the surfactant comprises dodecyl alcohol polyoxyethylene ether having an average of 9.5 ethoxylate groups.
28. The process of claim 18 wherein the surfactant comprises octylphenolpoly(ethyleneglycolether).