Real-time PCR quantifications in the presence of primers with different annealing abilities.

<p>Panel (A) shows amplification plots obtained for each condition; (B) shows the relative errors <i>Log(DNA_exp)</i>–<i>Log(DNA_obs)</i> obtained using <i>Cy0</i> and <i>Cy0</i> corrected following Eq. 10. PCRs were performed in the presence of an equal starting number of template molecules by using 500 nM of ND1R2 as reverse primers and different combinations of ND1F2 and ND1F5 as forward primers. Specifically, the PCRs were set as follows: 1) 500 nM ND1F2; 2) 375 nM ND1F2 and 125 nM ND1F5; 3) 250 nM ND1F2 and 250 nM ND1F5; 4) 200 nM ND1F2 and 300 nM ND1F5; 5) 150 nM ND1F2 and 350 nM ND1F5; 6) 125 nM ND1F2 and 375 nM ND1F5; 7) 100 nM ND1F2 and 400 nM ND1F5; 8) 50 nM ND1F2 and 450 nM ND1F5; and 9) 500 nM ND1F5. This experimental model offers the advantage of known PCR efficiencies of extreme conditions (condition 1 corresponds to HE while condition 9 corresponds to LE); hence, we were able to reproduce a gradient of mild decreasing efficiency ranging from 0.93 to 0.79. Black dots represent <i>Cy0</i> values while white dots correspond to <i>Cy0_corrected</i> values. Each symbol represents a mean of 4 runs. SOD detected all the runs obtained using [ND1F5]≥250 nM as outliers.