MiR-specific qPCR on synthetic templates with DNA primers. A The effect of primer concentration on Cq value of ssc-let-7d and ssc-miR-26a miR-specific qPCR assays. Real-time PCR assays were performed in parallel at three different concentrations (125, 250 and 500 nM) of the forward and of the reverse primers. B Amplification curves of an eight log10 dilution series of a synthetic ssc-let-7d template in the ssc-let-7d miR-specific qPCR assays. All samples contained a final concentration of 0.2 ng/μl salmon sperm DNA. C Extrapolation of Cq as function of the log10 of the number of templates for the same experiment as in B was a straight line (R2 = 0.9993) with slope of -3.341 (PCR efficiency = 99%) over eight log10 dilution of the template. D Melting curve analysis of the same experiment. No template control is labeled ntc. Melting curve analysis was performed from 60°C to 99°C.