Lysis and reverse transcription for mrna quantification

Abstract

The present invention is directed to a method for performing RT-PCR for amplifying a target RNA comprising the steps of a) lysis of a cellular sample which is supposed to contain the target RNA with a lysis buffer comprising between 0.2 M and 1 M Guanidine Thiocyanate, b) diluting the sample to an extend such that Guanidine Thiocyanate is present in a concentration of about 30 to 50 mM, e) reverse transcribing in the presence of a mixture of first strand cDNA synthesis primers, the mixture consisting of oligo dT primers and random primers, and d) subjecting the sample to multiple cycles of thermocycling protocol and monitoring amplification of the first strand cDNA in real time, characterized in that steps a) to c) are done in the same vessel.

1 . A method for performing a real time polymerase chain reaction (RT-PCR) for amplifying a target RNA comprising the steps of:
 lysing a biological sample which is supposed to contain said target RNA in a sample vessel with a lysis buffer comprising between 0.05 M and 1 M of Guanidine Thiocyanate,   diluting said sample to an extent such that said Guanidine Thiocyanate is present for a subsequent reverse transcription step in a concentration of about 30 to 50 mM in said sample vessel,   without any intermediate purification step, reverse transcribing said target RNA in the presence of a mixture of first strand cDNA synthesis primers into a first strand cDNA, said mixture consisting of primers hybridizing to a poly-A sequence or random primers or target specific primers in said sample vessel, and   amplifying said first strand cDNA by means of subjecting said sample to multiple cycles of a thermocycling protocol.   
     
     
         2 . The method according to  claim 1  wherein said amplification is monitored in real time. 
     
     
         3 . The method according to  claim 1  wherein said biological sample consists of not more than 1000 cells. 
     
     
         4 . The method according to  claim 1  wherein said biological sample consists of not more than 100 cells. 
     
     
         5 . The method according to  claim 1  wherein said biological sample consists of not more than a single cell. 
     
     
         6 . (canceled) 
     
     
         7 . The method according to  claim 1  wherein said lysis buffer comprises between about 0.2 and 0.5 M Guanidine Thiocyanate. 
     
     
         8 . (canceled) 
     
     
         9 . The method according to  claim 1  wherein the lysis step is performed in the presence of NP40 (octyl phenoxylpolyethoxylethanol) and wherein said non-ionic detergent during the reverse transcription step has a V/V of 0.5 to 2%. 
     
     
         10 . The method according to  claim 1  wherein the lysis step comprises the addition of a carbohydrate, which is preferably a sugar or a dextran. 
     
     
         11 . The method according to  claim 1  wherein the lysis step is performed for at least 5 minutes at ambient temperature or below ambient temperature. 
     
     
         12 . The method according to  claim 1  wherein the lysis step is performed for at least 5 minutes at a temperature between about 55° C. to 85° C., preferably in the presence of proteinase K. 
     
     
         13 . The method according to  claim 12  wherein between the lysis step and the dilution step or between the dilution step and the reverse transcription step, the sample is incubated for at least 5 minutes at a temperature between about 80° C. to 90° C. 
     
     
         14 . The method according to  claim 11  wherein the lysis step is performed in the presence of DNAse I or Shrimp Nuclease. 
     
     
         15 . The method according to  claim 14  wherein between the lysis step and the dilution step or between the dilution step and the reverse transcription step, the sample is incubated for at least 5 minutes at a temperature between about 80° C. to 90° C. 
     
     
         16 . The method according to  claim 11  wherein prior to the dilution step, the sample is frozen at temperatures between about −20° C. and −80° C. 
     
     
         17 . The method according to  claim 1  wherein said mixture of cDNA synthesis comprises primers hybridizing to a poly-A sequence and random primers. 
     
     
         18 . The method according to  claim 17  wherein primers hybridizing to a poly-A sequence and random primers are present in essentially equal molar amounts. 
     
     
         19 . The method according to  claim 17  wherein said primers hybridizing to a poly-A sequence and random primers are present in concentrations between 1 μM and 5 μM each. 
     
     
         20 . The method according to  claim 17  wherein said primers hybridizing to a poly-A sequence and random primers are present in concentrations of about 2.5 μM each.