Hi everyone!
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?
Thank you!