Reverse transcriptase
PCR (RT-
PCR) can be performed as a one or two step reaction. In one-step RT-
PCR, cDNA synthesis and subsequent
PCR amplification occur in the same tube. This saves on both time and reagents. In addition, since there is no intermediate cDNA purification step, one-step RT-
PCR is generally considered more sensitive and, therefore, better for detection of low abundance mRNA. About the only time you wouldn’t want to perform one-step RT-
PCR is if your RNA is from a rare or difficult-to-obtain source. In this case, you may opt to separate the RT-
PCR reaction into two steps by performing the intermediate cDNA purification; this will allow you to store some of that precious material for other experiments. Otherwise, one-step RT-
PCR is your ticket to fast, efficient and sensitive detection of gene products. For quantitative expression, of course, you’ll want to look to real-time RT-
PCR.
