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.
Sellner
L N
LN
Department of Pathology, Royal Perth Hospital, Perth, WA 6000, Australia.
Turbett
G R
GR
D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008545:Melanoma; D012334:RNA, Neoplasm / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D012334:RNA, Neoplasm; D004259:DNA-Directed DNA Polymerase