Since the early descriptions of the use of quantitative Real Time PCR, the technique has been adopted in almost every aspect of life science research and is increasingly used for clinical analysis. Over time protocols and strategies have been tried and tested, amended and developed such that there are currently several different approaches. Protocol variations are evident at each step of the RT-qPCR process, from sample acquisition to data analysis (e.g. sample QC, experimental design, assay design and validation, normalisation, biostatistical interpretation, reporting, etc). It is now apparent that these adaptations may result in differences in the final biological conclusion of the study.
Each step of the RT-qPCR will be discussed and the variability in resulting data that is due to different protocols will be illustrated.