Crude biological derivatives competent for nucleic acid detection

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.

1 . A method comprising:
 obtaining at least one biological unit containing RNA;   obtaining at least one catabolic enzyme;   preparing an admixture of the biological unit and the catabolic enzyme; and   incubating the admixture under conditions where the catabolic enzyme is active.   
     
     
         2 . The method of  claim 1 , further comprising obtaining at least two catabolic enzymes. 
     
     
         3 . The method of  claim 2 , wherein the at least two catabolic enzymes are a protease and a nuclease. 
     
     
         4 . The method of  claim 3 , wherein the protease is Proteinase K and the nuclease is DNase I. 
     
     
         5 . The method of  claim 1 , wherein the biological unit is a cell. 
     
     
         6 .- 7 . (canceled) 
     
     
         8 . The method of  claim 5 , wherein the cell is in obtained from a cell culture. 
     
     
         9 . The method of  claim 5 , wherein the cell is a prokaryotic cell. 
     
     
         10 . The method of  claim 5 , wherein the cell is a fungal cell. 
     
     
         11 . The method of  claim 5 , wherein the cell is a eukaryotic cell. 
     
     
         12 . The method of  claim 11 , wherein the cell is a human cell. 
     
     
         13 . The method of  claim 5 , wherein the biological unit is obtained from a subject. 
     
     
         14 . The method of  claim 5 , wherein the biological unit is obtained from a sample of body fluid. 
     
     
         15 . (canceled) 
     
     
         16 . The method of  claim 13 , wherein the biological unit is in a tissue sample. 
     
     
         17 . The method of  claim 1 , wherein the biological unit is a virus. 
     
     
         18 . The method of  claim 1 , wherein the catabolic enzyme is a protease. 
     
     
         19 .- 26 . (canceled) 
     
     
         27 . The method of  claim 1 , further comprising adding an RNase inhibitor to the admixture. 
     
     
         28 .- 34 . (canceled) 
     
     
         35 . The method of  claim 1 , further defined as a method for producing cDNA from one or more biological units and further comprising incubating the admixture with reverse transcriptase under conditions to promote reverse transcription. 
     
     
         36 . The method of  claim 35 , further comprising amplifying the products of the reverse transcription. 
     
     
         37 . The method of  claim 35 , further comprising incubating said admixture with a deoxyribonuclease prior to the reverse transcription reaction. 
     
     
         38 .- 40 . (canceled) 
     
     
         41 . A method for producing cDNA from one or more biological units comprising:
 obtaining at least one biological unit;   obtaining at least one catabolic enzyme;   preparing an admixture of the biological unit and the catabolic enzyme; and   incubating the admixture at a temperature where the catabolic enzyme is active and with reverse transcriptase under conditions to allow reverse transcription.   
     
     
         42 . The method of  claim 41 , further comprising obtaining at least two catabolic enzymes. 
     
     
         43 . The method of  claim 42 , wherein the at least two catabolic enzymes are a protease and a nuclease. 
     
     
         44 . (canceled) 
     
     
         45 . The method of  claim 41 , wherein the reverse transcriptase is added to the admixture after a time sufficient to allow the catabolic enzyme to function. 
     
     
         46 .- 55 . (canceled) 
     
     
         56 . The method of  claim 41 , further comprising amplifying the products of the reverse transcription. 
     
     
         57 . The method of  claim 41 , further comprising incubating said admixture with a deoxyribonuclease prior to the reverse transcription reaction. 
     
     
         58 . A kit for producing cDNA from a biological unit, comprising, in a suitable container:
 a buffer; and   a catabolic enzyme.   
     
     
         59 .- 63 . (canceled) 
     
     
         64 . A kit for producing cDNA from a biological unit comprising, in one or more suitable container(s):
 a biological unit lysis buffer;   a deoxyribonuclease;   an RNase inhibitor;   a reverse transcription buffer;   reverse transcriptase;   dNTPs; and   an Armored RNA® control.   
     
     
         65 .- 70 . (canceled)