Absolute Copy Number from the Statistics of the Quantification Cycle in Replicate Quantitative Polymerase Chain Reaction Experiments

作者：Tellinghuisen Joel^*; Spiess Andrej Nikolai

来源：Analytical Chemistry, 2015, 87(3): 1889-1895.

DOI：10.1021/acs.analchem.5b00077

The quantification cycle (C-q) is widely used for calibration in real-time quantitative polymerase chain reaction (qPCR), to estimate the initial amount, or copy number (N-0), of the target DNA. C-q may be defined several ways, including the cycle where the detected fluorescence achieves a prescribed threshold level. For all methods of defining C-q, the standard deviation from replicate experiments is typically much greater than the estimated standard errors from the least-squares fits used to obtain C-q. For moderate-to-large copy number (N-0 > 102), pipet volume uncertainty and variability in the amplification efficiency (E) likely account for most of the excess variance in C-q. For small N-0, the dispersion of C-q is determined by the Poisson statistics of N-0, which means that N-0 can be estimated directly from the variance of C-q. The estimation precision is determined by the statistical properties of chi(2), giving a relative standard deviation of similar to(2/n)(1/2), where n is the number of replicates, for example, a 20% standard deviation in N-0 from 50 replicates.

• 出版日期2015-2-3