Lysis and improved reverse transcription for mRNA quantification

摘要

Method for performing an RT-PCR for amplifying a target RNA comprising the steps of: a) lysis of a biological sample supposed to contain said target RNA in a sample vessel with a lysis buffer comprising between 0.05 M and 1 M guanidine thiocyanate, b) diluting said sample to the point where said guanidine thiocyanate is present during step c) at a concentration of between about 10 and 60 mM in said sample vessel, c) without any intermediate stage of purification, retrotranscribing said target RNA in the presence of a mixture of synthesis primers first strand cDNA in a first strand cDNA, consisting of said mixture primers hybridizing to a poly-a and / or random primers and / or specific primers of the target, in said sample vessel, d) amplifying said first strand cDNA by subjecting said sample to multiple cycles of a protocol thermocycled do