Nucleic acids | RNA identification and quantification via RT-qPCR

This chapter describes the identification and quantification of nucleic acids, in particular ribonucleic acid (RNA) using quantitative polymerase chain reaction (qPCR) combined with reverse transcription PCR (RT-qPCR). It summarizes various options and the connected problems in the quantitative workflow of mRNA and microRNA measurement, covering the initial RNA extraction to final data analysis. In detail the RNA isolation and their quality control, the reverse transcription, the reaction kinetics, and the quantification strategies are described and discussed. A further focus is set on various aspects in RT-qPCR data analysis, comparing absolute- versus relative-quantification approaches, the PCR efficiency correction, how to process the raw data, and the right normalization strategy according to the well-established MIQE guidelines is presented.