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Improved Strategies and Optimization of Calibration Models for Real-time PCR Absolute Quantification
SIVAGANESAN, M., R. A. HAUGLAND, E. C. CHERN, AND O. C. SHANKS. Improved Strategies and Optimization of Calibration Models for Real-time PCR Absolute Quantification. WATER RESEARCH. Elsevier Science Ltd, New York, NY, 44(16):4726-4735, (2010).
To inform the public.
Real-time PCR absolute quantification applications rely on the use of standard curves to make estimates of DNA target concentrations in unknown samples. Traditional absolute quantification approaches dictate that a standard curve must accompany each experimental run. However, the generation of a standard curve for each qPCR experiment set-up can be expensive and time consuming, especially for studies with large numbers of unknown samples. As a result, many researchers have adopted a master calibration strategy where a single curve is derived from DNA standard measurements generated from multiple instrument runs. However, a master curve can inflate uncertainty associated with intercept and slope parameters and decrease the accuracy of unknown sample DNA target concentration estimates. Here we report two alternative strategies termed pooled and mixed for the generation of calibration equations from absolute standard curves which can help reduce the cost and time of laboratory testing, as well as the uncertainty in calibration model parameter estimates.
Record Details:Record Type: DOCUMENT (JOURNAL/PEER REVIEWED JOURNAL)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL RISK MANAGEMENT RESEARCH LABORATORY
WATER SUPPLY AND WATER RESOURCES DIVISION
MICROBIAL CONTAMINANTS CONTROL BRANCH