1-step vs. 2-step Real Time PCR - pros and cons (Feb/05/2006 )

I've just started (Trying) to use real time PCR and have been using 2-step. It seems to me, that using 1 step would be beneficial (if I have enough RNA) because it cuts out the time (and contamination/ product loss) involved in the RT. However there seems to be some objection to that method both here and in the literature.

So I was just wondering why 2-step is preferred?

Mostly helps because you can use a housekeeping gene for normalization... in one step RT-PCR the sample is RT'd then PCR'd in the same tube--therefore all the sample is used for one PCR reaction--- RT efficiencies vary so you should normalize to houskeeping gene to compare samples...

(it should be noted that one step could be used to test two genes simultaneously in semi-quantitative PCR--this can be done using PCR with two primer sets on the same sample -- assuming you have optimized conditions and only want to look at one thing + housekeeping gene...)

doing two step just gives you more sample to work with, allows you to check more genes on the same sample etc..

Anyway, this is what I think...

HTH

It is case by case, I think.

In case of analyzing just one or two genes in large numbers of samples, for instance hundreds of samples, it would be better using one step for saving the time and energy.

While most of our research works, in fact, need analyzing many genes in very limited samples, sometimes required further check after a period, so the 2 step is more preferred. Advantage of 2 step is that you might not worry about RNA degradation since RT products is more stable in preservation than RNA. Furthermore, the 2 step is more reproducible than one step as mention by beccaf22 and many other papers [ For recent review: Biotechniques. 2005 Jul;39(1):75-85]

Thanks for the answers...

does this

that does not work with sybr green; it is for primer/probe sets

the reason is, sybr green measures amount of fluorescence, based on the amount of product...it would just look like tons of product was in the tube and you would not be able to differentiate relative amounts.