SeqrunqPCR efficiency from a standard curve
A qPCR assay that works well roughly doubles the product every cycle. How close it gets to a perfect doubling is its amplification efficiency, and you read it off the slope of a standard curve.
The standard curve
Run a dilution series of known template — say 10-fold steps — and plot the quantification cycle (Cq) against the log₁₀ of the starting quantity. The points fall on a line. Because each 10-fold dilution should shift Cq by a fixed number of cycles, the slope of that line encodes efficiency.
Slope to efficiency
amplification factor = 10^(−1 ÷ slope); efficiency = factor − 1
A slope of −3.322 gives 10^(1 ÷ 3.322) = 2.00 — a perfect doubling — so efficiency is 2.00 − 1 = 100%. That specific number comes from log₁₀(2) ≈ 0.301: a doubling every cycle means a 10-fold change takes 3.322 cycles. A steeper slope of −3.10 gives 2.10 → 110%.
The acceptable window
Most guidelines accept 90–110% efficiency (slope −3.58 to −3.10) with a standard-curve R² above 0.98. Inside that window, ΔΔCq relative quantification is valid. Outside it, the assumption that each cycle doubles the product breaks down and quantification is biased.
When efficiency looks wrong
Efficiency above 110% is usually an artefact — inhibitors carried through the dilution series, primer-dimers, or pipetting error making the slope too steep — not genuine super-doubling. Below 90% points to suboptimal primers, secondary structure, degraded template, or too much template inhibiting the reaction. Fixing primer design and cleaning up template are the usual remedies.