When you did your second experiment, you only had a 4 Ct difference between your highest and lowest DNA quantities, whereas your were spanning about 13 Ct in the first one. When you validate qPCR primers, you want to know if they give linear amplification over a wide range of DNA quantities / concentrations, so your first way to go (5 1-in-10 dilutions) was good; if you don't have enough dilutions of your template, you won't test extreme conditions. Secondly, if you get bad efficiency and/or r-square, it means that your primers are not optimal for linear amplification, so there is no way to go around. I don't really understand what you mean by "I tried to find good efficiency by combinating 5 datas that I have", but if you removed some values to increase either efficiency and R-square then that'll automatically impinge upon the other parameter... I would advise you to design, order and test a new pair of primers that work better... Good luck!

Hi

the best thing when you designed primers is to test them by an in silico PCR (you'll get access at the NCBI tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) or the UCSC one (http://genome.ucsc.edu/cgi-bin/hgPcr), with of course the right organism) before ordering them. now it's done, test the primers to see if the PCR will product an amplification and see the appropriate Tm.

in regards to what you said, cDNA is too concentrated (I don't know your reaction volume, I guess10-25ul) for all molecules to access the targets and therefore to perform reliable PCR, try less with appropriate primers.

fred

Hi,

The R^2 and efficiency are different, the R^2 is the coefficient of variance between replicates at a single concentration (how scattered they are), whereas the efficiency shows the slope of your standard curve.

It is possible that the R^2 value may be down to primer design (as suggested in the previous comments), but I have personally found that it generally comes down to the pipet you are using and how well maintained and calibrated it is.

An efficiency above 1 (100%) is normally an indication of the presence of inhibitors, whereas below 1 indicate pipetting error, poor primer design or bad reaction conditions.

So I would suggest first trying a different set of pipettes that have recently been calibrated and if you still have problems, then look at the primers.

Steve