How to do successful gene expression analysis using real-time PCR

Reverse transcription quantitative PCR (RT-qPCR) is considered today as the gold standard for accurate, sensitive and fast measurement of gene expression. Unfortunately, what many users fail to appreciate is that numerous critical issues in the workflow need to be addressed before biologically meaningful and trustworthy conclusions can be drawn. Here, we review the entire workflow from the planning and preparation phase, over the actual real-time PCR cycling experiments to data-analysis and reporting steps. This process can be captured with the appropriate acronym PCR: plan/prepare, cycle and report. The key message is that quality assurance and quality control are essential throughout the entire RT-qPCR workflow; from living cells, over extraction of nucleic acids, storage, various enzymatic steps such as DNase treatment, reverse transcription and PCR amplification, to data-analysis and finally reporting.