qPCR Efficiency Calculator (Standard Curve)
Free qPCR amplification efficiency calculator. Enter a serial dilution standard curve and get E = 10^(-1/slope) - 1, percent efficiency, R2 and MIQE pass/fail checks, plus the ΔCt-slope test that decides whether 2^-ΔΔCt is valid.
How it works
A serial dilution of template is amplified and the mean Cq of each dilution is regressed against log10 of the relative template amount by ordinary least squares. The slope of that line gives amplification efficiency as E = 10^(-1/slope) - 1: a perfectly doubling reaction has a slope of -3.32 and E = 100%. The fit also returns R2 and the dynamic range covered, and each is checked against the MIQE reporting thresholds — 90-110% efficiency, R2 of at least 0.98, and four or more points spanning three or more logs. When a reference gene curve is entered as well, the difference between the two slopes is the DeltaCt-slope test: below 0.1 the comparative 2^-DeltaDeltaCt method is valid, and above it the Pfaffl efficiency-corrected model should be used instead. All computation happens in the browser; Cq values are never uploaded.
Frequently asked questions
How do you calculate qPCR amplification efficiency from a standard curve?
Run a serial dilution of template — typically five 10-fold dilutions — and plot the mean Cq of each dilution against the base-10 logarithm of the relative template amount. Fit a straight line and read the slope. Efficiency is E = 10^(-1/slope) - 1, expressed as a percentage. A slope of -3.32 corresponds to 100% efficiency, meaning the product doubles every cycle.
What is an acceptable qPCR efficiency?
The MIQE guidelines treat 90-110% as acceptable, which corresponds to a standard-curve slope between -3.58 and -3.10, with an R2 of at least 0.98 across three or more logs of template. Values below 90% usually point to inhibitors carried over from extraction, degraded template, or primers with stable secondary structure. Values above 110% are almost never real amplification: they generally reflect primer-dimer or non-specific product, pipetting error in the dilution series, or a threshold set inside the baseline noise.
Why is my qPCR efficiency over 100%?
Amplification cannot exceed doubling per cycle, so an efficiency above 100% is a measurement artefact rather than a biological result. The usual causes are a non-specific product or primer-dimer inflating signal at the dilute end of the curve, inhibitors that suppress the concentrated dilutions more than the dilute ones, serial-dilution pipetting error, or a Cq threshold placed too low. Check the melt curve for a single peak and repeat the dilution series with fresh dilutions before accepting the number.
Do target and reference genes need the same efficiency for 2^-DeltaDeltaCt?
Yes. The comparative 2^-DeltaDeltaCt method assumes both amplicons amplify at close to the same efficiency. The standard test is to plot DeltaCt (target Cq minus reference Cq) against log10 template across the dilution series: if the absolute slope is below 0.1, the assumption holds. If it is larger, use the Pfaffl model, which corrects each target by its own measured efficiency, instead of assuming perfect doubling for both.
How many dilution points does an efficiency curve need?
MIQE recommends at least four dilution points spanning three or more orders of magnitude, each run in technical replicate, and reporting the slope, R2, efficiency and dynamic range alongside the result. Five 10-fold dilutions is the common practical choice. Points where the Cq approaches the no-template control should be excluded, since they sit outside the assay linear range and drag the slope.
Method reference
Bustin SA et al. (2009) Clin Chem 55(4):611-622 (MIQE guidelines); Pfaffl MW (2001) Nucleic Acids Res 29(9):e45.