need advice on efficiency - (Jun/22/2006 )

Hi all,

I've been doing several rounds of real time PCR and I've experienced some problems with the efficiency.
First of all, I can not figure out what effiency corresponds to in the PCR reaction.
Secondly, for exactly the same reaction : same RT, same primers, same setup but done in two separate days, the efficiency went from 0.9 which is very good to 0.5 which is poor. Has anybody ever experienced that trouble ?
Thirdly, if the effiency of one particular gene X reaction is 0.6 and the one for the housekeeping gene is 0.5, is that OK to go on with the gene X quantification anyway ?

I looking forward your reply...

David

oh gosh, you need to get that ironed out..those are way low


I was told by the ABI guru who coached me through my green period that you wanted efficiencies between 85% and 110%...that an efficiency of 100% (or 1.0) means that your PCR is giving you exactly one doubling per cycle. also, he said that for your results to be reproducible, efficiencies between one gene and the other need to be <10% if you're using standard curves, <5% if you're using ddCt. Otherwise it is very difficult to reproduce your results

I suspect this imay be part of your problem?