Crude biological derivatives competent for nucleic acid detection
View Patent ↗The invention relates to methods for the detection of a specific sequence of RNA in a cell or tissue sample. The invention also relates to methods to enzymatically manipulate the RNA in a crude cell lysate in a number of applications.
1. A method for producing cDNA from at least one biological unit containing RNA, wherein the biological unit is a cell obtained from cell culture, from a tissue sample or from a body fluid, comprising:
preparing an admixture comprising the biological unit, proteinase K and a deoxyribonuclease;
incubating the admixture under conditions where the proteinase K and deoxyribonuclease are active;
raising the temperature of the admixture containing the active proteinase K and deoxyribonuclease to 75 degrees C. to inactivate the proteinase K and the deoxyribonuclease;
incubating the admixture with at least one reverse transcriptase under conditions to allow reverse transcription to produce cDNA;
and
further comprising amplifying the cDNA by real-time PCR.
2. The method of claim 1 wherein the deoxyribonuclease comprises DNase 1.
3. The method of claim 1 , wherein the biological unit is a cell obtained from cell culture.
4. The method of claim 1 , wherein the biological unit is a cell obtained from a tissue sample.
5. The method of claim 1 , wherein the biological unit is a eukaryotic cell.
6. The method of claim 5 , wherein the eukaryotic cell is a human cell.
7. The method of claim 1 wherein the biological unit is a prokaryotic cell.
8. The method of claim 1 , wherein the biological unit is a fungal cell.
9. The method of claim 1 , wherein the period for inactivation of the proteinase K and deoxyribonuclease at 75° C. is 10 minutes.
10. A method for producing cDNA for quantification of RNA from at least one biological unit containing RNA, wherein the biological unit is a cell obtained from cell culture, from a tissue sample or from a body fluid, comprising:
preparing an admixture comprising the biological unit a deoxyribonuclease and at least one catabolic enzyme, wherein said enzyme is at least proteinase K;
incubating the admixture under conditions where the proteinase K and the deoxyribonuclease are active;
raising the temperature of the admixture to 75 degrees C. to inactivate the proteinase K and deoxyribonuclease;
incubating the admixture with at least one reverse transcriptase under conditions to allow reverse transcription to produce cDNA.
11. The method of claim 10 , wherein the biological unit is a cell obtained from cell culture.
12. The method of claim 11 , further comprising amplifying the cDNA by real-time PCR.
13. The method of claim 10 , wherein the biological unit is a cell obtained from a tissue sample.
14. The method of claim 10 , wherein the biological unit is a eukaryotic cell.
15. The method of claim 14 , wherein the eukaryotic cell is a human cell.
16. The method of claim 10 , wherein the biological unit is a prokaryotic cell.
17. The method of claim 10 , wherein the biological unit is a fungal cell.
18. The method of claim 10 , wherein the period for inactivation of the proteinase K and deoxyribonuclease at 75 degrees C. is 10 minutes.