Figure 3

Effect of amplification efficiency over the quantifications performed by different methods. (A) In-silico generated PCR data with initial template amount T₀ = 0.001 and different intrinsic amplification efficiencies (Ei) ranging from 0.65 to 0.972 analysed by different methods (see below). Data points represent the base 2 logarithm of the ratio between T₀ estimations from each simulated reaction and efficiency 0.8 ones vs. the amplification efficiency bias as mean ± SEM of triplicates. (B) Analysis of experimental results by different methods (see below). Bars represent the error of quantifications as mean ± SEM for triplicates. Bars marked with (*) are under-estimations, conversely, the rest of the bars are over-estimations. Method 6 under-estimated T₀ by 737%, note that it is out of scale in the graph. Data was analysed with the CT method assuming constant amplification efficiency equal to 1 {1}; with the CT method assuming constant amplification efficiency equal to 0.8 for in-silico data, or 0.855 for experimental data {2}; assuming constant amplification efficiency that was estimated from two threshold values {3} [15]; using the assumption-free analysis proposed by Ramakers et.al. {4} [13]; using the standardized determination of PCR efficiency from single reaction proposed by Tichopad et.al. {5} [16]; using the sigmoid model proposed by Liu et. al. {6} [17]; and with our model based real-time PCR analysis method (MoBPA) {7}.