One-Tube RT-PCR with Sequence-Specific Primers

From the very beginning of PCR technology (1 – 3 ), it was clear that the power of amplification could be used for the study of mRNA expression (4 ,5 ). This technique is now wldeiy used and many different protocols have been developed using either viral reverse transcriptases (RT) or exploiting the reverse transcriptase activity Inherent in many thermostable DNA polymerases (6 ). The main problem related to this technique when compared to other methods of RNA analysis is the primer design, which remains mamly emplrical. The normal approach to reduce nonspecific reactions is the use of nested primers which, however, further complicates the reaction scheme. Another posslbility is the use of a sequence-specific RT primer different from the PCR primers which, however, must be removed before PCR amplification.