Abstract
In the analysis of quantitative PCR (qPCR) data, the quantification cycle (Cq) indicates the position of the amplification curve with respect to the cycle axis. Because Cq is directly related to the starting concentration of the target, and the difference in Cq values is related to the starting concentration ratio, the only results of qPCR analysis reported are often Cq, ΔCq or ΔΔCq values. However, reporting of Cq values ignores the fact that Cq values may differ between runs and machines, and, therefore, cannot be compared between laboratories. Moreover, Cq values are highly dependent on the PCR efficiency, which differs between assays and may differ between samples. Interpreting reported Cq values, assuming a 100% efficient PCR, may lead to assumed gene expression ratios that are 100-fold off. This review describes how differences in quantification threshold setting, PCR efficiency, starting material, PCR artefacts, pipetting errors and sampling variation are at the origin of differences and variability in Cq values and discusses the limits to the interpretation of observed Cq values. These issues can be avoided by calculating efficiency-corrected starting concentrations per reaction. The reporting of gene expression ratios and fold difference between treatments can then easily be based on these starting concentrations.
Highlights
The quantitative polymerase chain rection is based on the real-time monitoring of the fluorescence increase per cycle during the amplification of DNA
We will describe and discuss how reported quantitative polymerase chain rection (qPCR) results are biased by ignoring the dependence of Cq on amplification efficiency, how the setting of the quantification threshold leads to differences and variability in the observed Cq values and how unavoidable pipetting errors and sampling variation as well as PCR-affecting contaminants hamper the interpretation of reported Cq values
This paper shows comprehensively that differences in usage of materials, qPCR machines, laboratory protocols, analysis procedures and factors affecting the PCR kinetics all lead to differences in the observed Cq values within and between experiments
Summary
The quantitative polymerase chain rection (qPCR) is based on the real-time monitoring of the fluorescence increase per cycle during the amplification of DNA. We will describe and discuss how reported qPCR results are biased by ignoring the dependence of Cq on amplification efficiency, how the setting of the quantification threshold leads to differences and variability in the observed Cq values and how unavoidable pipetting errors and sampling variation as well as PCR-affecting contaminants hamper the interpretation of reported Cq values. Taken together, these issues can all vary between samples, assays, plates and laboratories. The paper will focus on the clinical implications of the factors affecting the observed Cq and the quantitative and diagnostic interpretation of observed Cq values
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