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The distribution interval of calibration curves in real-time PCR was successfully predicted from the variance of the Ct values, and the predicted distribution interval was consistent with the interval of the actual calibration curves. Although the width of the 95% confidence interval at the lower end was about 1%, the copy number estimated from individual calibration curves was distributed over a wider range, and the 95% confidence interval of the estimates of 20 copies was +/-40%. The variances of the Ct values at 20 copies were significantly larger than those at higher copy numbers, and this large variation increased the distribution interval of the calibration curves. The variation, however, can be attributed to the probabilistic distribution of copy numbers in the measurement system, and is impossible to reduce by improvement of the analytical methodology. The 95% confidence interval of the calibration curves obtained after omitting 20 copies was decreased and the measurement precision was improved.

Factors Affecting the Variance of Copy Numbers in Real-time PCR as Estimated from Calibration Curves