Crude biological derivatives competent for nucleic acid detection

Abstract

The invention relates generally to the fields of making biological unit lysates or admixtures of body fluids and of RNA analysis. More specifically, it relates to direct methods for the detection of a specific sequence of RNA in a biological unit, for example a virus, cell or tissue sample, or a body fluid, for example saliva, sputum, blood plasma, etc. More generally, the invention may be used to enzymatically manipulate and protect the RNA in lysate or bodily fluids for a number of applications.

1 . A method comprising: 
 obtaining at least one biological unit containing RNA or sample comprising RNA not comprised in a biological unit;    obtaining a low pH buffer;    mixing the biological unit and the buffer to prepare a low pH lysate or mixing the sample and the buffer to prepare a low pH admixture; and    mixing at least a portion of the lysate or admixture with a composition comprising    enzyme using RNA as a substrate to form a reaction mixture.    
     
     
         2 . The method of  claim 1 , wherein the low pH lysate or admixture has a pH of from 0 to 6.  
     
     
         3 . The method of  claim 2 , wherein the low pH lysate or admixture has a pH of less than 5.  
     
     
         4 . The method of  claim 1 , wherein the enzyme is comprised in a buffer that adjusts the pH of the reaction mixture to a level suitable for enzyme function upon mixing.  
     
     
         5 . The method of  claim 4 , wherein the pH of the reaction mixture is between 7.0 and 9.5.  
     
     
         6 . The method of  claim 1 , wherein the buffer comprised a strong-weak acid.  
     
     
         7 . The method of  claim 6 , wherein the acid has a pKa of 1-4.  
     
     
         8 . The method of  claim 7 , wherein the strong-weak acid is arginine, glycine, or chloroacetic acid.  
     
     
         9 . The method of  claim 6 , wherein the buffer further comprises a detergent.  
     
     
         10 . The method of  claim 9 , wherein the detergent is a non-ionic detergent.  
     
     
         11 . The method of  claim 9 , wherein the detergent is an anionic detergent or cationic detergent.  
     
     
         12 . The method of  claim 1 , further defined as comprising mixing RNA from the lysate or admixture with a composition comprising reverse transcriptase to form a reverse transcriptase reaction mixture and incubating the reaction mixture under conditions resulting in a reverse transcription reaction.  
     
     
         13 . The method of  claim 12 , wherein the reverse transcriptase is comprised in a reverse transcriptase buffer that adjusts the pH of the reaction mixture to a level suitable for reverse transcriptase function upon mixing.  
     
     
         14 . The method of  claim 13 , wherein the pH of the reaction mixture is between 7.0 and 9.5.  
     
     
         15 . The method of  claim 14 , wherein the pH of the reaction mixture is between 8.0 and 8.4.  
     
     
         16 . The method of  claim 12 , wherein the lysate or admixture is not incubated with a proteinase K, pepsin, another protease, or DNase prior to mixing at least a portion of the lysate or admixture with the composition comprising reverse transcriptase.  
     
     
         17 . The method of  claim 12 , wherein the lysate or admixture is not incubated with pepsin prior to mixing at least a portion of the lysate or admixture with the composition comprising reverse transcriptase.  
     
     
         18 . The method of  claim 12 , wherein the lysate or admixture is not incubated with a DNA or protein precipitating agent prior to admixture with the composition comprising reverse transcriptase.  
     
     
         19 . The method of  claim 12 , wherein the RNA is not isolated from the lysate or admixture prior to mixing at least a portion of the lysate or admixture with the composition comprising reverse transcriptase.  
     
     
         20 . The method of  claim 12 , wherein RNA is isolated from the lysate or admixture prior to mixing the RNA with the composition comprising reverse transcriptase.  
     
     
         21 . The method of  claim 12 , further comprising amplifying at least one cDNA product of the reverse transcription reaction.  
     
     
         22 . The method of  claim 21 , wherein the amplification involves PCR.  
     
     
         23 . The method of  claim 12 , further comprising determining the presence of and/or quantity of an RNA in the biological unit or sample.  
     
     
         24 . The method of  claim 23 , further comprising admixing an RNA control with the reaction mixture or the at least a portion of the lysate or admixture prior to reverse transcription.  
     
     
         25 . The method of  claim 23 , further defined as a method of determining whether or not an siRNA with which the biological unit or organism from which the sample is obtained has been contacted has altered the concentration of one or more RNA in the biological unit or body.  
     
     
         26 . The method of  claim 25 , further comprising comparing the presence of and/or quantity of cDNA products from the biological unit or organism contacted with the siRNA with cDNA products obtained from a biological unit or organism not contacted with an siRNA or contacted with a negative control siRNA.  
     
     
         27 . The method of  claim 23 , further defined as a method of determining whether or not a compound with which the biological unit or organism from which the sample is obtained has been contacted has altered the concentration of one or more RNA in the biological unit or sample.  
     
     
         28 . The method of  claim 27 , further comprising comparing the presence of and/or quantity of cDNA products from the biological unit or organism contacted with the compound with cDNA products obtained from a biological unit or organism not contacted with the compound or contacted with a control.  
     
     
         29 . The method of  claim 23 , further comprising employing a labeled probe or intercalating dye to determine the presence of and/or quantify the RNA.  
     
     
         30 . The method of  claim 1 , further comprising detecting one or more protein in the lysate or admixture.  
     
     
         31 . The method of  claim 30 , wherein the protein is detected in an antibody-based assay.  
     
     
         32 . The method of  claim 31 , wherein the antibody-based assay comprises immunoblotting, ELISA, or immunoprecipitation.  
     
     
         33 . The method of  claim 1 , further defined as amplifying RNA from the lysate or sample.  
     
     
         34 . The method of  claim 33 , further comprising analyzing the amplified RNA in a microarray analysis.  
     
     
         35 . The method of  claim 1 , further comprising making a low pH biological unit lysate.  
     
     
         36 . The method of  claim 35 , wherein the biological unit is a cell.  
     
     
         37 . The method of  claim 36 , wherein the cell is a prokaryotic cell.  
     
     
         38 . The method of  claim 36 , wherein the cell is a fungal cell.  
     
     
         39 . The method of  claim 36 , wherein the cell is a eukaryotic cell.  
     
     
         40 . The method of  claim 39 , wherein the cell is a human cell.  
     
     
         41 . The method of  claim 36 , wherein the biological unit is obtained from a subject.  
     
     
         42 . The method of  claim 41 , wherein the biological unit is obtained from a sample of body fluid.  
     
     
         43 . The method of  claim 42 , wherein the body fluid is saliva, sputum, plasma, serum, whole blood, cerebral spinal fluid, fecal material, or urine.  
     
     
         44 . The method of  claim 41 , wherein the biological unit is in a tissue sample.  
     
     
         45 . The method of  claim 36 , wherein the biological unit is a cell from a cell culture.  
     
     
         46 . The method of  claim 35 , wherein the biological unit is a virus.  
     
     
         47 . The method of  claim 1 , further defined as comprising making an admixture of sample comprising RNA not comprised in a biological unit and the buffer.  
     
     
         48 . The method of  claim 47 , wherein the sample is a body fluid.  
     
     
         49 . The method of  claim 47 , wherein the body fluid is saliva, sputum, whole blood, plasma, serum, cerebral spinal fluid, fecal material or urine.  
     
     
         50 . The method of  claim 47 , wherein the sample comprises partially purified or purified RNA.  
     
     
         51 . The method of  claim 1 , wherein the RNA remains substantially intact at ambient temperature.  
     
     
         52 . The method of  claim 1 , wherein the RNA remains substantially intact from RNA degradation if the lysate is stored for 8 hours at ambient temperature.  
     
     
         53 . The method of  claim 1 , wherein lysis or admixing occurs between 15° C. and 37° C.  
     
     
         54 . The method of  claim 1 , wherein the lysis or admixing occurs at ambient temperature.  
     
     
         55 . The method of  claim 1 , further comprising adding an RNase inhibitor to the lysate or admixture.  
     
     
         56 . A method of assaying RNA comprising: 
 obtaining at least one biological unit containing RNA or sample comprising RNA not comprised in a biological unit;    obtaining a low pH buffer;    mixing the biological unit and the buffer to prepare a low pH lysate or mixing the sample and the buffer to prepare an admixture; and    mixing at least a portion of the lysate or admixture with a composition comprising reverse transcriptase to form a reverse transcriptase reaction mixture and incubating the reaction mixture under conditions resulting in a reverse transcription reaction to prepare cDNA.    
     
     
         57 . The method of  claim 56 , further comprising amplifying cDNA products of the reverse transcription reaction.  
     
     
         58 . The method of  claim 56 , further comprising determining the presence of and/or quantity of an RNA in the biological unit or sample.  
     
     
         59 . The method of  claim 58 , further comprising employing a labeled probe or intercalating dye to determine the presence of and/or quantify the RNA.  
     
     
         60 . A kit for assaying RNA in a biological unit or sample comprising, in one or more suitable container(s): 
 a low pH buffer, a high pH buffer, or a buffer that precipitates RNA;    a reverse transcription buffer;    reverse transcriptase; and    dNTPs.    
     
     
         61 . The kit of  claim 60 , further comprising an RNA control.  
     
     
         62 . The kit of  claim 60 , further comprising a thermostable DNA polymerase.  
     
     
         63 . The kit of  claim 60 , further comprising an RNase inhibitor.  
     
     
         64 . The kit of  claim 60 , further defined as comprising a low pH buffer.  
     
     
         65 . The kit of  claim 60 , further defined as comprising a high pH buffer.  
     
     
         66 . The kit of  claim 60 , further defined as comprising a buffer that precipitates RNA.  
     
     
         67 . A method comprising: 
 obtaining at least one biological unit containing RNA or sample comprising RNA not comprised in a biological unit;    obtaining a high pH buffer;    mixing the biological unit and the buffer to prepare a high pH lysate or mixing the sample and the buffer to prepare a high pH admixture; and    mixing at least a portion of the lysate or admixture with a composition comprising an enzyme using RNA as a substrate to form a reaction mixture.    
     
     
         68 . The method of  claim 67 , wherein the lysate or admixture has a pH of from 9 to 14.  
     
     
         69 . The method of  claim 68 , wherein the lysate or admixture has a pH of greater than or equal to 11 and less than 14.  
     
     
         70 . The method of  claim 69 , further comprising mixing at least a portion of the lysate or admixture with a composition comprising reverse transcriptase to form a reverse transcriptase reaction mixture and incubating the reaction mixture under conditions resulting in a reverse transcription reaction.  
     
     
         71 . The method of  claim 70 , wherein the reverse transcriptase is comprised in a reverse transcriptase buffer that adjusts the pH of the reaction mixture to a level suitable for reverse transcriptase function.  
     
     
         72 . The method of  claim 71 , wherein the pH of the reaction mixture is between 7.0 and 9.5.  
     
     
         73 . The method of  claim 72 , wherein the pH of the reaction mixture is between 8.0 and 8.4.  
     
     
         74 . A method comprising: 
 obtaining at least one biological unit containing RNA or sample comprising RNA not comprised in a biological unit;    obtaining a buffer;    mixing the biological unit and the buffer to prepare a low pH lysate or mixing the sample and the buffer to prepare an admixture, wherein the buffer precipitates RNA in the lysate or admixture; and    detecting and/or quantifying RNA in the biological unit or sample.    
     
     
         75 . The method of  claim 74 , wherein the buffer is a low pH buffer.  
     
     
         76 . The method of  claim 74 , wherein the buffer is a high pH buffer.  
     
     
         77 . The method of  claim 74 , wherein the buffer comprises a detergent.  
     
     
         78 . The method of  claim 77 , wherein the buffer comprises a non-ionic detergent.  
     
     
         79 . The method of  claim 77 , wherein the detergent comprises an anionic or cationic detergent.