RT-PCR — Delta Ct Relative Quantification
Analyze RT-qPCR gene expression with 2⁻ΔΔCt (Livak), Pfaffl, and 2⁻ΔCt methods. Based on RQdeltaCT methodology.
Formula: Assumes 100% efficiency. Most common method.
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📖 Delta Ct Methods Reference
2⁻ΔΔCt (Livak)
Most widely used. Assumes ~100% PCR efficiency for both target and reference genes. ΔCt = Ct(target) − Ct(ref), then ΔΔCt = ΔCt − mean(ΔCt_control).
Pfaffl Method
Corrects for different PCR efficiencies. Ratio = E_target^(ΔCt_target) / E_ref^(ΔCt_ref). Use when amplification efficiencies differ between genes.
2⁻ΔCt (Simple)
Normalizes gene expression to reference gene only. Expression = 2^(−ΔCt). Useful for comparing expression levels across samples without a calibrator.
References: Livak & Schmittgen (2001) Methods 25:402–408 · Pfaffl (2001) Nucleic Acids Res 29:e45 · Zalewski & Bogucka-Kocka (2025) Sci Rep 15:29762