Practical approach to quantification of mRNA abundance using RT‑qPCR, normalization of experimental data and MIQE

Abstract

Reverse transcription and quantitative polymerase chain reaction (RT-qPCR) has become the most common method for characterizing expression patterns of individual mRNAs due to a large dynamic range of linear quantification, high speed, sensitivity, resolution and cost-effectiveness. However, the complexity of the protocol, variability of reagents, an inconsistent quality of biological samples, and the absence of standardized methods of data quantification may produce inconsistent results. In an effort to to standardize ithe procedure and assure high reliability of data, the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines was defined and further extended by Prof. Bustin and colleagues (2004). These guidelines have been followed by the development of an XML-based real-time PCR data markup language (RDML) and a RDML data base for consistent reporting of RT-qPCR data. Here we follow the RT‑qPCR procedure step by step in compliance with MIQE requirements, local facilities and resources and our own experience in application of RT-qPCR methodology.