Compute relative gene expression using the 2^-ΔΔCt method. Import RDML files or paste Ct values. Efficiency correction, outlier detection, and MIQE checklist. Data never leaves your browser.
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The ΔΔCt method normalizes gene expression in two steps:
1. ΔCt = Ct(target gene) − Ct(reference gene) — normalizes for RNA input 2. ΔΔCt = ΔCt(treatment) − mean(ΔCt(control)) — normalizes to baseline 3. Fold Change = 2^(−ΔΔCt) — converts to linear scale
The method assumes equal amplification efficiency (~100%) for all genes. When this assumption is violated, use the Pfaffl efficiency-corrected method.
The MIQE guidelines (Bustin et al., 2009) define the minimum information needed for reviewers to assess qPCR experiments. Key requirements:
• RNA quality: Report RIN values, A260/280, A260/230 • Primer validation: Show specificity (melt curve) and efficiency (standard curve) • Reference genes: Validate stability across conditions • Analysis: State quantification method and software used
The interactive MIQE checklist in this tool helps you document compliance systematically.
