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What are the advantages and disadvantages of one step vs. two step RT-PCR?
In the one-step method, the reverse transcriptase is included in the same tube as the PCR. The two-step method involves creating cDNA in a separate reverse transcription reaction and then adding some of this cDNA to the PCR. There are advantages and disadvantages to each of these methods.
One-step RT-PCR is attractive due to convenience and reduced opportunities for contamination, but is usually less sensitive, more complicated to troubleshoot, and does not provide a stock of cDNA for future use.
Two-step RT-PCR is highly sensitive, potentially more efficient, and allows for stocking of the cDNA for future use. However, this method is more time consuming and can require more optimization.
To learn more, read Starting with RNA—one-step or two-step RT-qPCR?