Primer validation/efficiency by qRT-PCR in five simple steps

You have now amplified and measured the Ct of your serial cDNA dilutions... Now what? 😅 To measure the efficiency of your primer couple, you have probably been recommended the serial dilution method💧 🌡️ This method involves making dilutions of your typical control sample (e.g., 1 in 4 , 1 in 16 , 1 in 64 , 1 in 256 , 1 in 1024 ) and mixing it with your typical Master mix and your favorite primer concentration. This way you can confirm in silico whether the amplification pattern of your primers in successively diluted samples fits a trend line. How to measure the efficiency of your primer couple using the serial dilution method💦 1) If you measured Ct values from at least 3 replicates of each dilution, calculate the Ct average of the 3 replicates for each dilution . 2) Consider your 1 in 4 sample as a hypothetical cDNA concentration value of 100 (100%), since that will be the highest sample concentration point. The remainder will be: 1 in 16 (25%), 1 in 64 (6.25%), 1 in 256 ...