real time RT-PCR, 1-step vs. 2-step method - (Feb/13/2009 )

Hello,

could someone with a bit more experience than I have (I´m naive to this method yet) describe me the pros and cons of 1-step versus 2-step real time reverse transcription PCR. Meaning that when it would be better to do the reverse transcription separately and when in one and the same tube along with the PCR. The 1-step method is of course tempting but there must be some disadvantages too. Thank you, I appreciate the effort!

Hi,

if you use a one-step reaction you simply can not make replicates of your sample in you real-time PCR. Means you can not distinguish if occouring variance is due to your RT-reaction or your real-time PCR.
Another reason might be that you have to split up sample after the RT-reaction if you use SYBR-green and want to amplify more than one gen. Otherwise you should not compare these genes with each other, as the RT-efficiencies might vary between different reactions.

hope it helped,

Jan

In my opinion, one-step has its eligibility only in diagnostic or forensic laboratories were cross contamination or pipetting errors would have profound consequences. For research use I would always go for two-step. it is far more convenient.