Hi!
I notice that reverse transcriptases can be used for both cDNA library construction with template switching (eg. surescript II etc.), and downstream qPCR. To quality-control my library construction, I would like to do a qPCR of focused gene with the first strand cDNA, but reverse transcripted with template switching. However, even with the same reverse transcriptase, will the activation of template switching, as well as the addition of adaptor-UMI before poly T in primer?
Thank you!
Your question isn't terribly clear. Could you perhaps expand on that last bit, because at present it isn't actually a question.
If you're asking "can you use qPCR on cDNA reverse transcribed using a TSO-system?" then...yes. Yes you can.
Most conventional cDNA synthesis only makes first strand sequence, because you can use that for qPCR just fine: it's ssDNA initially, but the first round simply converts the target of interest to dsDNA, and after that it's PCR as per usual.
If you use a template switch system to make dsDNA, then...you just can PCR that as you would any other cDNA, except now the first round doesn't make your target into dsDNA (coz you've already done that) and so you'll typically measure your target about ~1 cycle earlier (not exactly, because TSO-mediated double stranded cDNA synthesis isn't 100% efficient).
But yeah, you can use it in qPCR.
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