How is qPCR efficiency calculated?

Efficiency comes from the slope of a line through Ct versus log10 of template amount. The fold change per cycle is 10 raised to the power of minus one over the slope, and efficiency percentage is that value minus one, times one hundred.

What slope means 100 percent efficiency?

A slope of about minus 3.32 corresponds to 100 percent efficiency, meaning the amount of product doubles every cycle. Slopes steeper than minus 3.6 or shallower than minus 3.1 fall outside the generally accepted 90 to 110 percent efficiency band.

Why does template go on the x-axis as a log?

Across a serial dilution the template spans several orders of magnitude, so plotting its base-10 logarithm linearises the relationship with Ct. The regression then has a constant slope from which efficiency is read directly.

What R-squared is acceptable?

An R-squared of 0.98 or higher shows the standards fall tightly on the regression line, indicating consistent pipetting and amplification. A lower value points to pipetting error, poor dilution accuracy, or a standard that should be excluded.

What if efficiency is above 100 percent?

Efficiency above 100 percent is physically impossible and usually signals a problem, most often inhibitors or pipetting error in the more concentrated standards that delay their Ct. Re-run the curve with fresh, accurately prepared dilutions to confirm.

qPCR Efficiency Calculator from Standard Curve

qPCR efficiency tells you whether your assay amplifies cleanly enough to trust its quantification. This calculator fits a line to your Ct-versus-log(template) standard curve and reports the efficiency, fold-per-cycle, and R-squared, with a pass or fail against the usual acceptance window.

How it works

The points are fit by ordinary least-squares linear regression of Ct against the base-10 logarithm of template amount. Efficiency is read from the slope:

E (fold per cycle) = 10^(−1 / slope)
efficiency %        = (E − 1) × 100

A perfectly doubling reaction has a slope of −3.322 and efficiency of 100 percent (E = 2). R-squared is computed from the regression residuals as 1 − SS_res / SS_tot, measuring how tightly the standards sit on the line.

Tips and notes

A well-behaved assay shows efficiency between 90 and 110 percent (slope between −3.6 and −3.1) and R-squared of at least 0.98. Run your standards as a clean serial dilution spanning four to six orders of magnitude in duplicate, and drop any single point that is an obvious pipetting outlier before reporting. An efficiency above 100 percent almost always means inhibitors or pipetting error in the concentrated standards, not faster-than-physical amplification.