What this calculator does
Amplification efficiency describes how close a qPCR reaction comes to doubling its target every cycle. It is calculated from the slope of a standard curve of Ct against log concentration, as 10 raised to the power of −1 over the slope, minus 1.
Perfect doubling gives a slope of −3.322, which is −log₁₀(2), and an efficiency of 100%. The accepted working range is roughly 90 to 110%, corresponding to slopes between about −3.6 and −3.1. Outside that, relative quantification comparing two targets starts producing results that are wrong rather than merely imprecise.
The formula
Efficiency is 10 to the power of minus one divided by the slope, minus 1, expressed as a percentage. The amplification factor is that efficiency plus 1, so a 100% efficient reaction has a factor of 2 and genuinely doubles each cycle. The ideal slope of −3.322 is shown alongside as the reference the measured slope is being judged against.
| Term | Meaning |
|---|---|
| Standard curve | Ct plotted against the log of template concentration across a dilution series. |
| Slope | The gradient of that line, negative because higher concentration gives lower Ct. |
| −3.322 | The slope corresponding to perfect doubling, equal to minus the base-ten log of 2. |
| Amplification factor | How much the target multiplies per cycle: 2.0 at 100% efficiency. |
The inputs explained
| Field | What to enter |
|---|---|
| Standard curve slope | The slope of the standard curve, which will be negative. A typical acceptable value falls between about −3.6 and −3.1. |
When to use it
Validating a new assay
Efficiency within the accepted range is a standard requirement before an assay is used for quantification, and it is usually reported alongside the R² of the curve.
Comparing target and reference genes
The comparative Ct method assumes both amplify at similar efficiency. Where they differ materially, the method gives biased results and an efficiency-corrected calculation is needed.
Diagnosing a poor reaction
Low efficiency usually points to inhibitors, suboptimal primers or a poor dilution series. Above 100% generally means something is wrong with the standards rather than that the reaction is unusually good.
Worked examples
Every figure in the tables below is produced by this page’s own calculator at build time, so the numbers and the tool always agree. Select any row to load that scenario.
What efficiency does each standard curve slope give?
A range of standard curve slopes and the efficiency each implies.
| Slope | Amplification efficiency | Amplification factor per cycle (1+E) | Ideal slope at 100% efficiency |
|---|---|---|---|
| -3.10 | 110.2% | 2.102 | -3.322 |
| -3.32 | 100.1% | 2.001 | -3.322 |
| -3.58 | 90.3% | 1.903 | -3.322 |
| -3.90 | 80.5% | 1.805 | -3.322 |
Questions
What is a good qPCR efficiency?
Between 90 and 110% is the usual acceptance range, corresponding to slopes from about −3.6 to −3.1. Within that, comparative quantification methods behave reliably. Outside it, the assumption that targets amplify equivalently starts to break down.
Why is the ideal slope −3.322?
Because perfect doubling means a tenfold change in template shifts Ct by log₂(10), which is about 3.32 cycles. The slope is negative because more template gives an earlier Ct. The figure is simply minus the base-ten logarithm of 2, inverted.
What does an efficiency above 100% mean?
Almost always a problem with the standards rather than a reaction that amplifies faster than doubling, which is physically impossible. Common causes are pipetting error in the dilution series, inhibitors carried through at higher concentrations, or primer-dimer contributing signal at the dilute end.
Does efficiency need to be identical between targets?
For the standard comparative Ct method, close enough that the difference does not matter, conventionally within 5%. Where efficiencies differ more than that, an efficiency-corrected model such as the Pfaffl method should be used instead, since the simple method assumes they match.
For converting concentration to copy number for standards, see the DNA copy number calculator. For annealing temperature, see the PCR annealing temperature calculator.