The efficiency of the reaction can be calculated by the following equation: E = 10(-1/slope) –1. The efficiency of the PCR should be 90-110% meaning doubling of the amplicon at each cycle. This corresponds to a slope of -3.1 to -3.6 in the Ct vs log-template amount standard curve (see Agilent Efficiency Calculator from the Slope). In order to obtain accurate and reproducible results, reactions should have efficiency as close to 100% as possible (e.g., two-fold increase of amplicon at each cycle), and in any case, efficiency should be similar for both target and reference (normalizer, calibrator, endogenous control, internal control). A number of variables can affect the efficiency of the PCR. These factors can include length of the amplicon, presence of inhibitors, secondary structure and primer design. Although valid data can be obtained that fall outside of the efficiency range, if it is < 0.90, the quantitative real-time PCR should be further optimized or alternative amplicons designed. If efficiency is found > 110%, try running a standard curve experiment with a minimum of 3 replicates and a minimum of 5 logs of template concentration and repeat the calculation. Efficiency of your reaction can be calculated using the online program CAmpER (Calculation of Amplification Efficiencies for RT-PCR). See Efficiency Determination and Standard Curve for more on efficiency.