Quantitative Real-Time RT-PCR

Quantitative real-time PCR is a sensitive tool for quantitative expression analysis of RNA isolated from clinical samples and is increasingly being utilized in novel clinical diagnostic assays. It is an essential tool for the detection of degraded RNA, as that extracted from formalin-fixed paraffin-embedded (FFPE) tissues. Although it has unquestionable potential for expression analysis in archival case studies, there are still some biological and technical problems associated with its use, which can be partly overcome by a standardization of the protocols. The first part of this chapter provides a detailed description of the most commonly used real-time-based chemistries, SybrGreen and TaqMan, focusing especially on their advantages when applied in FFPE material. Specific suggestions have been included in order to improve the reliability of the expression results. The second part of the chapter is dedicated to the description of general guidelines that should be followed to optimize the design of quantitative real-time PCR assays. They have been adapted to RNA isolated from FFPE but should always be taken into account in any gene expression analysis to ensure its accuracy and reproducibility.