Quantitative polymerase chain reaction: a framework for improving the quality of results and estimating uncertainty of measurement

Abstract

International comparability of quantitative polymerase chain reaction (qPCR) measurement results is critical for applied fields within industry, healthcare and forensics and may be achieved through traceability to the International System of Units, the SI. This process requires determining the accuracy of the quantitative result by estimation of the measurement uncertainty (MU). Here we present a relatively simple process for estimating MU for quantitative DNA measurements produced by qPCR. A worked example is given in which the qPCR method has been divided into four parts: DNA extraction; preparation of calibration curves; qPCR set-up and data analysis. A framework is provided in comprehensive tables that allow the identification of most potential sources of variation and bias. We show that each source can be either eliminated from the measurement process or captured in the MU data. The necessary data for MU estimation are relatively easy to gather but the combined uncertainty is only representative if all sources of variation have been incorporated. Although qPCR is given as an example, the principles apply to other nucleic acid measurements.