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qPCR Absolute Quantification Calculator

Quantify unknown samples from their Ct values against a standard curve of known quantities, with the curve's efficiency and a flag for any unknown outside the standards.

Formula

Ct=mlog⁡10(Q)+bC_t = m \log_{10}(Q) + b
Q=10 (Ct−b)/mQ = 10^{\,(C_t - b)/m}
QQ
amount in the unit of the standards, for example copies
m, bm,\ b
the standard curve's fit

How it works

Absolute quantification compares an unknown's Ct with a curve made from standards of known amount, such as a plasmid or synthetic fragment whose copy number was calculated from its mass and length. The standard curve is fitted by least squares, and each unknown's Ct is converted back to a quantity by inverting it.

The result is only valid inside the range of the standards. An unknown whose Ct lies beyond the highest or lowest standard is extrapolated, and is flagged. The accuracy of the answer is limited by the accuracy of the standards themselves, including their measured concentration and whether they behave like the real samples.

Worked example

The same six standards as in the efficiency example, with unknowns at Ct 20.0, 27.4 and 35.0.

  1. The curve is Ct = −3.354 × log₁₀(quantity) + 38.69.
  2. Ct 20.0 gives 10^((20.0 − 38.69) / −3.354) = 3.74 × 10⁵ copies.
  3. Ct 27.4 gives 2,329 copies; Ct 35.0 gives 12.6 copies, which is beyond the lowest standard (Ct 32.0) and flagged as extrapolated.

Two unknowns are quantified within the standards; the third is an extrapolation and should be re-run more concentrated.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • Standards and unknowns amplify with the same efficiency, which is not guaranteed if the matrix differs.
  • The standards' quantities are accurate.
  • Ct values are means of technical replicates.

Common mistakes

  • Quantifying an unknown whose Ct is outside the standard range.
  • Using a standard curve from one plate on another run without re-checking it.
  • Forgetting the dilution factor and the volume of template to get from copies per reaction to copies per sample.

Related tools

qPCR Efficiency CalculatorAmplification efficiency from a dilution series or a slopeOpen calculatorDNA Copy Number CalculatorNumber of copies in a mass of plasmid, amplicon or genomeOpen calculatorCalibration Curve Calculator (Standard Curve)Linear fit, residuals and an unknown with its uncertaintyOpen calculatorqPCR Replicate Ct CalculatorMean, SD and fold spread of replicate Ct values, with flagsOpen calculator

Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.