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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.