Crude biological derivatives competent for nucleic acid detection
AssigneeApplied Biosystems, LLC
InventorsBrittan Pasloske, Quoc Hoang
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Abstract
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.
Claims17
A method for producing cDNA and amplification thereof, 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 inactivate the proteinase K and deoxyribonuclease to produce an inactivated admixture;
- and
- carrying out one-step reverse transcription-PCR on the inactivated admixture.
A method for producing cDNA and amplification thereof 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 catabolic 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 inactivate the proteinase K and deoxyribonuclease to produce an inactivated admixture;
- and
- carrying out one-step reverse transcription-PCR on the inactivated admixture.
Dependencies are inferred from the claim text.