One step vs. two step RT-PCR?
In the one-step RT-PCR method, the reverse transcriptase is included in the same tube as the PCR. The two-step RT-PCR 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 RT-PCR methods.
Advantages of one-step RT-PCR
: It's attractive due to convenience and reduced opportunities for contamination,
Cons of one-step RT-PCR
: Usually less sensitive, more complicated to troubleshoot, and does not provide a stock of cDNA for future use.
Advantages of two-step RT-PCR:
Highly sensitive, potentially more efficient, and allows for stocking of the cDNA for future use.
Disadvantages of two-step RT-PCR:
More time consuming and can require more optimization.
To learn more, read Starting with RNA—one-step or two-step RT-qPCR