Efficiency comes from the slope of the standard curve
Run a dilution series (usually 10-fold), read the Ct at each dilution, and plot Ct against log10 of the concentration. The line's slope feeds the formula above; multiply E by 100 for a percentage. A slope of −3.322 gives exactly 100% — the point where the target doubles every cycle. The slope is negative because more starting DNA crosses the threshold at a lower Ct.
What each slope means
| Slope | Efficiency | Verdict |
|---|---|---|
| −3.10 | 110% | High edge of acceptable |
| −3.32 | 100% | Ideal (perfect doubling) |
| −3.58 | 90% | Low edge of acceptable |
| −3.90 | ~80% | Poor — optimize the assay |
| −2.90 | ~121% | Too steep — likely error |
Aim for a slope between −3.1 and −3.6, with R² above 0.98 so the line actually fits your points. A poor R² makes the efficiency number meaningless.
Why efficiency matters for your results
- Ct doubling. At 100% efficiency the product doubles each cycle, so a difference of 3.32 cycles equals a 10-fold difference in starting material.
- Delta-delta Ct assumption. The common 2−ΔΔCt method only holds when target and reference genes both run at ~100% (within about 5%). If they differ, use an efficiency-corrected model instead.
- One curve per assay. Efficiency shifts with primers, polymerase, buffer and template quality, so build a fresh standard curve for each gene and ideally each run.
Common questions
What is a good qPCR efficiency?
90 to 110% is the accepted range, and 95 to 105% is ideal. Inside that band a slope near -3.32 means your assay roughly doubles product each cycle, which is what the delta-delta Ct method assumes.
Why is my efficiency over 100%?
True efficiency cannot exceed 100%, so a reading above ~110% points to a problem: inaccurate serial dilutions, pipetting error, PCR inhibitors carried through, or picking Ct values from a non-linear part of the curve. Remake the dilution series and rerun.
What slope gives 100% efficiency?
A slope of -3.322 on a Ct versus log10(concentration) plot. It comes from -1 divided by log10(2), the slope at which the target doubles exactly once per cycle.
Can I get efficiency without a standard curve?
Yes, with the two-point delta-Ct method if you have two dilutions, but it is less reliable than a full 4 to 6 point standard curve. Each gene or assay needs its own curve.
