Absolute Copy Number from the Statistics of the Quantification Cycle in Replicate Quantitative Polymerase Chain Reaction Experiments
2015
Tellinghuisen, Joel | Spiess, Andrej-Nikolai
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 > 10²), 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 χ², 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.
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