RT-PCR, why two PCR steps?
Posted 28 July 2004 - 06:06 AM
I'm new to RT-PCR to measure siRNA knockdown efficiency. I looked at several protocols and found that some protocols suggest two distinct PCR steps with different primer pairs (different length of amplified product) after reverse transcription while other protocols only suggest one single PCR step.
Could somebody please help me out by letting me know why these two steps should be necessary or what the advantages of both versions are?
Thanks a lot!
Posted 28 July 2004 - 07:58 AM
It's up to personal preference.
One step RT-PCR has been around for a few years, before that, two-step RT-PCR was the standard.
They don't have much difference in terms of the final results but differ in the process. One step RT-PCR does RT and PCR in one tube (containing both reverse transcriptase and taq) starting from total RNA; while two step RT-PCR finishes this in two reactions by first converting RNA to cDNA and then amplifying from cDNA template.
If your RNA is precious and you want to study many genes, you can choose two step RT-PCR so that you will have enough cDNA for multiple amplfication. In your case you said you want to do siRNA screening which may involve only one gene, you can go one step, because it's fast.
BTW, I just ordered for the first time a one step kit yesterday for my RNAi experiment.
Hope that helps.
Posted 28 July 2004 - 05:02 PM
I'm not sure if I understood your question right: is it that there is a reverse transcription step and then 2 PCRs, or as pcrman suggests the whole thing is together in one step, or a single RT and single PCR?
If it is RT then 2 PCRs it's called nested PCR (the second lot of primers is within the first lot, giving smaller product). It is used for rare products (withcraft, no product could appear in first pcr, but will turn up in second) or when you want to be a bit more specific with your product (smaller chances of amplifying irrelevant sequences), or specific applications, such as mutagenesis.
I would try a single PCR for your siRNA work, and if it works stick with it, if not then try a 2step reaction