Determination of primer efficiency equivalency. A linear regression curve was fit to the plot of the log cDNA dilution vs the ΔC_t, for IR-A (A), IR-B (B), and IGF1R (C). The data represent a minimum of four different qRT-PCR experiments, with each primer concentration plated in triplicate. Each point is the mean of the triplicate values from one qRT-PCR experiment. ΔC_t was calculated as the C_t of the target gene minus the C_t of β-actin, the endogenous control. The y-axis of each graph is a close, compacted scale. The spread of values at each cDNA concentration is minimal and within the expected range. The slope of each curve is calculated to determine whether the reaction efficiencies of the target and endogenous control genes are similar. If the absolute value of the slope of the linear regression is close to zero, then the reaction efficiencies of the target and endogenous control genes are similar and the ΔΔC_t method of calculation can be applied.