Semi-quantitative real-time PCR for pain research

In this chapter we cover what we have found to be a "best practice" for real-time polymerase chain reaction (PCR) for relative mRNA quantification. We describe our techniques for tissue-sample collection and freezing, sample handling for quick and reproducible extraction of total RNA, first strand cDNA synthesis, real-time PCR amplification, and template dilution and storage for PCR. We offer our insights on intron-spanning primer design for genes (when applicable), effective primer selection vs reaction optimization, and relative quantification and sample normalization using housekeeping genes. Comments are also provided on the choice of PCR reagents including fluorescent probes, prevention of PCR "carry over," and on the practical aspects of real-time PCR theory and interpretation.