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qPCR Efficiency Calculator (Standard Curve Slope)

Calculate qPCR primer amplification efficiency from a dilution-series standard curve or a slope: E = 10^(−1/slope) − 1, with slope, R², intercept and the 90–110% acceptance check.

Input

Dilution factor between points

Ct — most concentrated

Ct — dilution 2

Ct — dilution 3

Ct — dilution 4

Ct — dilution 5

Slope (Ct vs log10 quantity)

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How it works

x = log10(relative quantity) of each dilution, y = Ct · slope = Σ(x − x̄)(y − ȳ) ÷ Σ(x − x̄)² · amplification factor = 10^(−1/slope) · efficiency (%) = (10^(−1/slope) − 1) × 100 · a slope of −3.32 = 100%

If PCR product exactly doubles each cycle, a 10-fold dilution delays the Ct by log2(10) = 3.32 cycles, so a plot of Ct against log10 of the input amount has a slope of −3.32 and the efficiency is 100%. Run a serial dilution of template (five points of a 10-fold or 2-fold series, in replicate), fit the line, and convert its slope. The MIQE guidelines expect efficiencies of 90–110% with R² above about 0.98; values above 100% usually signal inhibition in the concentrated samples or primer dimers, not super-efficient PCR.

Worked example

A 10-fold series gives Ct values of 15.12, 18.41, 21.83, 25.14 and 28.49. The fitted slope is −3.347 with R² 1.0000, so the amplification factor is 10^(1/3.347) = 1.990 and the efficiency is 99.0% — acceptable.

Sources: Bustin SA et al. The MIQE guidelines. Clin Chem 2009;55:611–22 · Thermo Fisher — Efficiency of real-time PCR

Frequently asked questions

What is a good qPCR efficiency?

Between 90% and 110% (slope between about −3.58 and −3.10), with R² above 0.98. Efficiencies of target and reference genes should be within roughly 5–10% of each other to use the 2^−ΔΔCt method.

Why is my efficiency above 100%?

Most often PCR inhibitors in the most concentrated samples (they delay those Ct values and flatten the curve), primer-dimer signal in SYBR Green assays, or pipetting errors in the dilution series. Drop the most concentrated point and recheck.

How do I calculate efficiency from a 2-fold dilution series?

Enter 2 as the dilution factor. With perfect efficiency each 2-fold dilution raises the Ct by 1 cycle, so the slope against log10 quantity is still −3.32.

How many dilution points do I need?

At least three, but five or more points spanning the expected Ct range, each in triplicate, give a much more reliable slope.

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This calculator is for educational purposes. Double-check important results before acting on them.