Scenario B: qPCR curves with similar threshold cycles can have different f-values. PCR amplification of the S27a transcript on the same RNA in two different duplicates, with the second duplicate set (blue and green line) obtained with only 70% of the polymerase concentration as in the first set (red and black line). The threshold cycle obtained from the second derivative maximum method was within 13.6 ± 0.3 cycles. Both insets depict the parameter estimates for 'f' and 'e' for the fitted curves, showing that these two parameters are highly correlated. Black circles indicate experimental measurements.