Primer concentration is quantitative Real-time PCR - (Mar/11/2008 )
I have been trying to optimize an absolute quantification real-time PCR reaction for a while… While trying to find out the best concentrations for each primer, I have obtained weird results (or at least that I cannot understand very well). I have tried different primers concentrations combinations (100nM, 200nM, 300nM, 400nM, 500nM) with two standards (the second was a 1:10 dilution of the first). The lower Cts appeared for F primer at of 400nM and R primer 200nM or 400nM, suggesting that the reaction is more efficient for these concentrations. However, when I look to the efficiency, it is much higher is the F primer concentration is 100nM. So, my question is what is the rational for this? Why the efficiency seems higher with lower concentration of primers, but Cts are higher?
Have you checked the melting curves for each primer concentration set? One explanation would be that at higher primer concentrations what you are amplifying is primer dimers, therefore lowering the Ct values...