If I am doing a qPCR experiment looking at gene expression between a wild-type and mutant and I plan on doing 3 biological replicates what is the best way to factor in primer efficiency? Would you perform standard curves on cDNA combined from all three replicates? Or perform a standard curve on each biological replicate separately?
To define efficiency of a primer set in qPCR, you could use a sample, with an high expression of your gene and perform a serial dilution of this sample. I do 2 dilutions to obtain 100%, 10% and 1%. You will obtain a standard curve with your efficiency of qPCR.
Do the same thing with the reference gene and the target gene.
I would perform standard curve of GAPDH at different molar ratios of each biological replicates separately.
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