qPCR Pfaffl calculator — Quantifies relative expression with PCR-efficiency correction via the Pfaffl method. Formula: Ratio = (E_target^ΔCt_target) / (E_ref^ΔCt_ref). Efficiency E = 10^(-1/slope) is auto-derived from standard-curve slope. Also compares against the simpler ΔΔCt (Livak) method assuming E=2.

(1) Get corrected fold-change when target/reference PCR efficiencies differ (Livak ΔΔCt is biased when E≠2) (2) Accurate relative expression comparison: Treated vs Control (3) Auto-convert standard-curve slope to efficiency (E = 10^(-1/slope)) (4) Cross-validate qPCR results with both Pfaffl and ΔΔCt simultaneously

① Target gene • Mean Ct (Treated) • Mean Ct (Control) • Efficiency E (1.0–2.0) or standard-curve slope ② Reference gene (GAPDH, β-actin, etc.) • Mean Ct (Treated/Control) • Efficiency E or slope ③ Auto-output: • Pfaffl ratio (relative fold-change) • Livak ΔΔCt ratio (assumes E=2) • Side-by-side comparison Standard-curve slope ≈ -3.32 → E ≈ 2.00 (100% efficiency). slope ≈ -3.58 → E ≈ 1.90.

Example) IL-6 expression Treated vs Control, GAPDH normalization Target (IL-6): Ct_treated = 22.5, Ct_control = 25.8, slope = -3.32 → E = 2.00 Reference (GAPDH): Ct_treated = 18.4, Ct_control = 18.6, slope = -3.45 → E ≈ 1.95 ΔCt_target = 25.8 − 22.5 = 3.3 → E_target^3.3 ≈ 9.85 ΔCt_ref = 18.6 − 18.4 = 0.2 → E_ref^0.2 ≈ 1.14 → Pfaffl ratio = 9.85 / 1.14 = 8.6× upregulated → Livak ΔΔCt 2^((25.8-22.5)-(18.6-18.4)) = 2^3.1 = 8.6× (close match here)