qPCR efficiency calculator
Maintained by the Talindrew team · Last updated
qPCR amplification efficiency comes from the slope of a standard curve (Ct plotted against log₁₀ of template amount): E = 10^(−1/slope) − 1. A slope of −3.32 means perfect doubling each cycle (100%); the MIQE guidelines accept efficiencies between 90% and 110% with R² ≥ 0.98.
Result
E = 10^(−1/slope) − 1 from a Ct vs log₁₀(template) standard curve. A slope of −3.32 is exactly 100% (doubling each cycle); MIQE guidelines accept 90–110% with R² ≥ 0.98.
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What it looks like
Formula
E = 10^(−1/slope) − 1 · % efficiency = 100 × E · slope −3.32 ⇒ 100%
Source: Pfaffl, NAR 2001; MIQE guidelines (Bustin et al., Clin Chem 2009)
Worked example
Given: Standard curve slope −3.58, R² 0.998
- 1.E = 10^(−1/−3.58) − 1 = 10^0.2793 − 1 = 1.903 − 1 = 0.903.
- 2.Efficiency = 90.3%: at the lower edge of the acceptable window.
- 3.Amplification factor per cycle = 1.90 instead of the ideal 2.00.
Slope −3.58 → 90.3% efficiency
How the calculation flows
Units & constants
| Ideal slope | −3.32 (100%, exact doubling) |
|---|---|
| Acceptable (MIQE) | 90–110% efficiency, R² ≥ 0.98 |
| Slope −3.1 | 110%: usually inhibition or pipetting artefact |
| Slope −3.6 | 90%: borderline acceptable |
| Axes convention | Ct on y, log₁₀(amount) on x: flipped axes flip the slope sign |
| Price | Free |
Why does efficiency matter for ΔΔCt analysis?
Relative quantification assumes both target and reference amplify with the same, near-100% efficiency; each cycle then corresponds to an exact factor of two. At 90% efficiency a 3-Ct difference is a 6.9-fold change, not 8-fold, a 15% error that compounds with larger Ct spans. When efficiencies differ between assays, use efficiency-corrected models (Pfaffl) instead of ΔΔCt.
What causes efficiency above 110%?
Apparent super-doubling is always an artefact: PCR inhibitors carried into the concentrated standards (efficiency 'improves' as they dilute out), co-amplifying primer-dimers, or pipetting bias across the series. Clean up the template, redesign primers, or build the curve from a fresh dilution series.
What causes low efficiency?
Common culprits, roughly in order of frequency:
- Suboptimal primer design: secondary structure or mispriming (check with the Tm and self-complementarity calculators).
- Amplicon too long: keep qPCR products at 70–200 bp.
- Annealing temperature too high for the primer pair.
- Degraded or inaccurately quantified standards.
Frequently asked questions
About −3.44: solving 10^(−1/m) = 1.95 gives m = −1/log₁₀(1.95) = −3.44. Between the ideal −3.32 and the 90% boundary at −3.59.
No: some instruments report the amplification factor per cycle (E + 1), where 2.0 is perfect. 1.9 on that scale is 90% efficiency on this page's scale. Check which convention your instrument uses before comparing.
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