Abstract
BackgroundIn real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. However, potential differences in the amplification performance of plasmid DNA compared to genomic DNA standards are often ignored in calibration calculations and in some cases impossible to characterize. A flexible statistical method that can account for uncertainty between plasmid and genomic DNA targets, replicate testing, and experiment-to-experiment variability is needed to estimate calibration curve parameters such as intercept and slope. Here we report the use of a Bayesian approach to generate calibration curves for the enumeration of target DNA from genomic DNA samples using absolute plasmid DNA standards.ResultsInstead of the two traditional methods (classical and inverse), a Monte Carlo Markov Chain (MCMC) estimation was used to generate single, master, and modified calibration curves. The mean and the percentiles of the posterior distribution were used as point and interval estimates of unknown parameters such as intercepts, slopes and DNA concentrations. The software WinBUGS was used to perform all simulations and to generate the posterior distributions of all the unknown parameters of interest.ConclusionThe Bayesian approach defined in this study allowed for the estimation of DNA concentrations from environmental samples using absolute standard curves generated by real-time qPCR. The approach accounted for uncertainty from multiple sources such as experiment-to-experiment variation, variability between replicate measurements, as well as uncertainty introduced when employing calibration curves generated from absolute plasmid DNA standards.
Highlights
In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration
We report the use of a Bayesian approach to generate calibration curves for the enumeration of target DNA from genomic DNA samples using absolute plasmid DNA standards
We employed a Bayesian approach for the estimation of DNA concentrations from environmental samples using absolute standard curves generated by real-time quantitative PCR (qPCR)
Summary
In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. We report the use of a Bayesian approach to generate calibration curves for the enumeration of target DNA from genomic DNA samples using absolute plasmid DNA standards. The goal for many real-time quantitative PCR (qPCR) assays with clinical, forensic, or environmental applications is to develop a standardized method that can be implemented on an inter-laboratory scale. BMC Bioinformatics 2008, 9:120 http://www.biomedcentral.com/1471-2105/9/120 uct production as the reaction occurs. Under ideal conditions these products accumulate exponentially in the reactions, i.e. their quantities double with each thermal cycle. The CT value can be used to estimate the initial concentration of a DNA target from an unknown sample
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