Comparison of Three RT-PCR Methods

Various approaches can now be taken for amplification of RNA transcripts using the polymerase chain reaction (PCR). Here, we compare three such methods: (i) uncoupled reverse transcription (RT)-PCR (using separate reactions for cDNA synthesis and PCR), (ii) continuous RT-PCR (in which RT and DNA amplification occur in an uninterrupted reaction) using either a single enzyme for both RT and DNA amplification or (iii) using two enzymes, one for each task. We have found that the continuous two-enzyme RT-PCR method is the most sensitive, followed by the uncoupled RT-PCR and then the continuous single-enzyme method. The continuous methods require less sample handling than the uncoupled method, and hence are less labor-intensive and less prone to contamination. The continuous single-enzyme method is the most expensive to perform in terms of reagents due to the quantity of DNA polymerase required; however, it does have advantages over the two enzyme methods in that the use of a thermostable enzyme for RT can alleviate certain problems by allowing RT to occur at higher temperatures than those tolerable by viral reverse transcriptases.