Absolute copy number from the statistics of the quantification cycle in replicate quantitative polymerase chain...

The quantification cycle (Cq) is widely used for calibration in real-time quantitative polymerase chain reaction (qPCR), to estimate the initial amount, or copy number (N0), of the target DNA. Cq may be defined several ways, including the cycle where the detected fluorescence achieves a prescribed threshold level. For all methods of defining Cq, the standard deviation from replicate experiments is typically much greater than the estimated standard errors from the least-squares fits used to obtain Cq. For moderate-to-large copy number (N0 > 10(2)), pipet volume uncertainty and variability in the amplification efficiency (E) likely account for most of the excess variance in Cq. For small N0, the dispersion of Cq is determined by the Poisson statistics of N0, which means that N0 can be estimated directly from the variance of Cq. The estimation precision is determined by the statistical properties of χ(2), giving a relative standard deviation of ∼(2/n)(1/2), where n is the number of replicates, for example, a 20% standard deviation in N0 from 50 replicates.