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Frequently asked questions

Our Scientific Applications Support team has assembled a list of frequently asked questions to help you find answers quickly. Filter using one or more categories to focus on specific topics, or use the search bar to perform a text search.

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.

One-Step RT-PCR

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.

Two-Step RT-PCR

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?