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 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 protease activity, and a surfactant,
and
admixing the lysate with a stop mixture at substantially the same temperature as the contacting step to form a stopped mixture.
2. A process for preparing a sample containing DNA for in situ analysis of DNA or a surrogate thereof, the process comprising:
contacting the sample containing DNA with a lysis mixture at 16° C. to 28° C. for a time and under conditions to produce a lysate wherein the lysis mixture comprises a polypeptide having protease activity, and a surfactant;
and
admixing the lysate with a stop mixture at substantially the same temperature as the contacting step.
3. The process of claim 1 wherein the lysis mixture further comprises a polypeptide having deoxyribonuclease activity.
4. The process of claim 3 further comprising contacting the stopped mixture with reagents for reverse transcription to form a first amplification mixture.
5. The process of claim 4 wherein the stopped mixture comprises up to 45% of reverse transcription reaction volume.
6. The process of claim 4 wherein the stopped mixture comprises up to 65% of reverse transcription reaction volume.
7. The process of claim 3 further comprising contacting the stopped mixture with reagents for reverse transcription and polymerase chain reaction.
8. The process of claim 7 wherein the stopped mixture comprises up to 45% of reverse transcription and polymerase chain reaction volume.
9. The process of claim 7 wherein the stopped mixture comprises up to 65 % of reverse transcription and polymerase chain reaction volume.
10. The process of claim 2 further comprising contacting the stopped mixture with reagents for polymerase chain reaction.
11. The process of claim 10 wherein the stopped mixture comprises up to 45% of polymerase chain reaction volume.
12. The process of claim 10 wherein the stopped mixture comprises up to 65% of polymerase chain reaction volume.
13. The process of claim 1 wherein the contacting and admixing are carried out at 16° C. to 28° C.
14. The process of claim 1 wherein the contacting and admixing are carried out at ambient temperature.
15. The process of claim 1 wherein the sample comprises a cell or cell culture.
16. The process of claim 1 wherein the sample comprises a tissue sample or a sample comprising a virus.
17. The process of claim 2 wherein the sample comprises a cell or cell culture.
18. The process of claim 2 wherein the sample comprises a tissue sample or a sample comprising a virus.
19. The process of claim 3 wherein the polypeptide having deoxyribonuclease activity is stabilized by calcium cations and the lysis mixture further comprises a calcium salt.
20. The process of claim 1 wherein the polypeptide having protease activity comprises proteinase K or an enzymatically active mutant or variant thereof.
21. The process of claim 2 wherein the polypeptide having protease activity comprises proteinase K or an enzymatically active mutant or variant thereof.