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

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• 作者：Joel Tellinghuisen ; Andrej-Nikolai Spiess
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：February 3, 2015
• 年：2015
• 卷：87
• 期：3
• 页码：1889-1895
• 全文大小：424K
• ISSN：1520-6882

文摘

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 (N₀), 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 (N₀ > 10²), pipet volume uncertainty and variability in the amplification efficiency (E) likely account for most of the excess variance in C_q. For small N₀, the dispersion of C_q is determined by the Poisson statistics of N₀, which means that N₀ can be estimated directly from the variance of C_q. The estimation precision is determined by the statistical properties of 蠂², giving a relative standard deviation of 鈭?2/n)^1/2, where n is the number of replicates, for example, a 20% standard deviation in N₀ from 50 replicates.