simulation of between repeat variability in real time pcr reactions模拟之间的重复变化实时pcr反应.pdfVIP

Simulation of between Repeat Variability in Real Time PCR Reactions 1,2 2 1,3 2 Antoon Lievens *, Stefan Van Aelst , Marc Van den Bulcke , Els Goetghebeur 1 Platform for Molecular Biology and Biotechnology, Scientific Institute of Public Health, Brussels, Belgium, 2 Department of Applied Mathematics and Computer Science, Ghent University, Gent, Belgium, 3 Molecular Biology and Genomics Unit, European Commission - Joint Research Centre, Institute for Health and Consumer Protection, Ispra, Italy Abstract While many decisions rely on real time quantitative PCR (qPCR) analysis few attempts have hitherto been made to quantify bounds of precision accounting for the various sources of variation involved in the measurement process. Besides influences of more obvious factors such as camera noise and pipetting variation, changing efficiencies within and between reactions affect PCR results to a degree which is not fully recognized. Here, we develop a statistical framework that models measurement error and other sources of variation as they contribute to fluorescence observations during the amplification process and to derived parameter estimates. Evaluation of reproducibility is then based on simulations capable of generating realistic variation patterns. To this end, we start from a relatively simple statistical model for the evolution of efficiency in a single PCR reaction and introduce additional error components, one at a time, to arrive at stochastic data generation capable of simulating the variation patterns witnessed in repeated reactions (technical repeats). Most of the variation in Cq values was adequately captured by the statistical model