If we design a primer set against a target intended for their use in one-step qRT-PCR (reverse transcription followed by cDNA to DNA amplification in the same reaction tube), can that same primer set be used for the same target in a two-step PCR process (RNA to cDNA synthesis, and cDNA to DNA amplification in separate tubes)? Or in other words, can a primer pair be used commonly for both one-step and two-step PCR experiments? Do we need to design primers exclusively for one-step and two-step PCR experiments?
Will there be any primer-target binding issues?
Thank you.
There is no difference in the primers you'd use. "one-step" vs. "two-step" is just a difference in how you get the cDNA.
This allows for efficient and rapid amplification of the target but also increases the risk of contamination of the reverse transcriptase enzyme.
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