How to correct RT-qPCR Cq data for amplification efficiency — a method demonstration
The 2^(−ΔΔCt) shortcut assumes every assay doubles its product each cycle (100% efficiency). When a target and reference assay differ in efficiency, an uncorrected ratio is biased. Efficiency correction rescales each Cq to a common 100%-efficiency basis (the Pfaffl principle) before quantities are compared.
Efficiency (%) = (-1 + 10^(-1 / slope)) * 100 E = 1 + Efficiency(%) / 100 Ct(100%) = Cq * log2(E) Quantity = 100 * 2^( mean(std100 Ct(100%)) - sample Ct(100%) ) Normalized ratio = Target quantity / Reference quantity
| Group | Rep 1 | Rep 2 | Rep 3 | Mean | SD |
|---|---|---|---|---|---|
| A | 0.446925 | 0.495239 | 0.436762 | 0.459642 | 0.031244 |
| B | 0.821351 | 0.893508 | 0.580211 | 0.765024 | 0.164068 |
| C | 0.221490 | 0.299057 | 0.675234 | 0.398594 | 0.242697 |
| Assay | R² | Efficiency |
|---|---|---|
| Target | 0.999754 | 94.92% |
| Reference | 0.999960 | 98.84% |