Efficiency = (10^(-1/slope) − 1) × 100%
PCR efficiency measures how well template DNA is copied each cycle. Perfect efficiency (100%) means exact doubling each cycle — slope of -3.322 on a log10 dilution standard curve. Efficiency = (10^(-1/slope) − 1) × 100%. Acceptable range: 90-110% with R² > 0.98. A slope of -3.1 = 110%, -3.32 = 100%, -3.6 = 90%. Efficiency <90% suggests: primer dimers, secondary structures, long amplicons, inhibitors, or degraded template. Efficiency >110% suggests: pipetting errors or inhibition at high concentrations. For relative quantification (ΔΔCt), target and reference gene efficiencies should be within 5% of each other. Always validate efficiency with a 5-point, 10-fold dilution series.