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 kit for preparation of a sample containing RNA for in situ detection of RNA or a surrogate thereof, comprising:
lysis mixture components comprising:
a polypeptide having protease activity,
a surfactant comprising octylphenol ethoxylate having an average of 9.5 ethoxylate groups, octylphenol ethoxylate having an average of 7.5 ethoxylate groups, octylphenolpoly(ethyleneglycolether), dodecyl alcohol polyoxyethylene ether, or a combination thereof, and
a polypeptide having deoxyribonuclease activity,
wherein the lysis solution components are substantially free of a cation chelator;
and
stop mixture components comprising: a cation chelator, and a peptide containing inhibitor of the polypeptide having protease activity.
2. The kit of claim 1 further comprising a reverse primer, a reverse transcriptase buffer and dNTPs.
3. The kit of claim 2 further comprising a reverse transcriptase.
4. The kit of claim 3 further comprising a DNA polymerase.
5. The kit of claim 1 wherein the polypeptide having deoxyribonuclease activity is stabilized by calcium cations and the lysis mixture components further comprise a calcium salt.
6. The kit of claim 1 wherein the polypeptide having protease activity comprises proteinase K or an enzymatically active mutant or variant thereof.
7. The kit of claim 5 wherein the polypeptide having deoxyribonuclease activity comprises DNase I or an enzymatically active mutant or variant thereof.
8. The kit of claim 1 wherein the surfactant comprises octylphenol ethoxylate having an average of 7.5 ethoxylate groups.
9. The kit of claim 1 wherein the inhibitor of the polypeptide having protease activity comprises SEQ ID NO:1 or an active mutant or analog thereof.
10. The kit of claim 1 wherein the cation chelator of the stop mixture comprises EGTA.
11. The kit of claim 1 further comprising a ribonuclease inhibitor.
12. The kit of claim 11 wherein the ribonuclease inhibitor comprises placental ribonuclease inhibitor protein.
13. The kit of claim 4 further comprising an intercalating dye.
14. The kit of claim 4 further comprising a label having an emission spectra between and including 300 nm to 750 nm.
15. 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 ambient temperature and under conditions and for a time to produce a lysate,
stopping lysis 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.
16. The process of claim 15 wherein the sample contains RNA and reagents for nucleic acid polymerization comprise a reverse transcriptase.
17. The process of claim 16 wherein the reagents for nucleic acid polymerization comprise a reverse transcriptase and a DNA polymerase.
18. The process of claim 15 wherein the lysis mixture comprises a ribonuclease inhibitor.
19. The process of claim 15 wherein the lysis mixture comprises 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.