Abstract
BackgroundPCR has the potential to detect and precisely quantify specific DNA sequences, but it is not yet often used as a fully quantitative method. A number of data collection and processing strategies have been described for the implementation of quantitative PCR. However, they can be experimentally cumbersome, their relative performances have not been evaluated systematically, and they often remain poorly validated statistically and/or experimentally. In this study, we evaluated the performance of known methods, and compared them with newly developed data processing strategies in terms of resolution, precision and robustness.ResultsOur results indicate that simple methods that do not rely on the estimation of the efficiency of the PCR amplification may provide reproducible and sensitive data, but that they do not quantify DNA with precision. Other evaluated methods based on sigmoidal or exponential curve fitting were generally of both poor resolution and precision. A statistical analysis of the parameters that influence efficiency indicated that it depends mostly on the selected amplicon and to a lesser extent on the particular biological sample analyzed. Thus, we devised various strategies based on individual or averaged efficiency values, which were used to assess the regulated expression of several genes in response to a growth factor.ConclusionOverall, qPCR data analysis methods differ significantly in their performance, and this analysis identifies methods that provide DNA quantification estimates of high precision, robustness and reliability. These methods allow reliable estimations of relative expression ratio of two-fold or higher, and our analysis provides an estimation of the number of biological samples that have to be analyzed to achieve a given precision.
Highlights
PCR has the potential to detect and precisely quantify specific DNA sequences, but it is not yet often used as a fully quantitative method
Quantitative PCR is used widely to detect and quantify specific DNA sequences in scientific fields that range from fundamental biology to biotechnology and forensic sciences
Quantitative PCR usually relies on the comparison of distinct samples, for instance the comparison of a biological sample with a standard curve of known initial concentration, when absolute quantification is required [16], or the comparison of the expression of a gene to an internal standard when relative expression is needed
Summary
PCR has the potential to detect and precisely quantify specific DNA sequences, but it is not yet often used as a fully quantitative method. A number of data collection and processing strategies have been described for the implementation of quantitative PCR. They can be experimentally cumbersome, their relative performances have not been evaluated systematically, and they often remain poorly validated statistically and/or experimentally. Quantitative PCR is used widely to detect and quantify specific DNA sequences in scientific fields that range from fundamental biology to biotechnology and forensic sciences. The treatment of laboratory measurements is often fairly empirical in nature, and the validity or reproducibility of the assay remains usually poorly characterized from an experimental and/or theoretical basis. QPCR results have been questioned [2,3], with the consequence that semi-quantitative methods (e.g. end-point PCR) remain widely used
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