One-step versus two-step qRT-PCR - (Jan/11/2007 )
Hi, I was wondering if someone could explain the pro's and cons to either doing quantitative real time PCR as one step from mRNA (mRNA -> cDNA -> cDNA product amplification using gene specific primers) versus two step with cDNA being reversibly transcribed from mRNA using poly(t) primers. Then using the cDNA product with gene specific primers for product amplification. Is one method preferable to the other?
case by case, I think
if you want analysis a large number sample, one step is more efficient and cost effective
while you need analysis several gene in the limited sample, two step is better
there are also many literature for this online or referebce journal
"Real-time PCR for mRNA quantification" BioTechniques 39:75-85 (July 2005).
i do 2 step...
i like being able to do just plane RT PCR on my samples, so i can actually see there is a difference, rather than rely on the real time machine.
Commercial one step RT-PCR reactions are usually set up to maximize the PCR yields. If that's good enough, perform the one step protocol.
If you want maximum reverse transcriptase efficiency and maximum PCR efficiency, perform the two step reaction.
Generally speaking, two-step RT-PCR is more repeatable, and cDNA is more stable to keep than RNA sample.
I like the one step because there is less pipeting which I feel contributes significantly to the varaition in some samples.