High‐Throughput RT‐PCR for Small‐Molecule Screening Assays

Abstract Quantitative measurement of the levels of mRNA expression via real‐time reverse transcription polymerase chain reaction (RT‐PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used somewhat less frequently to measure the changes in gene expression due to perturbagens such as small molecules or siRNA. The availability of new instrumentation for liquid handling and real‐time PCR analysis, as well as the commercial availability of start‐to‐finish kits for RT‐PCR, has enabled the use of this method for high‐throughput small‐molecule screening on a scale comparable to traditional high‐throughput screening (HTS) assays. This protocol focuses on the special considerations necessary for using quantitative RT‐PCR as a primary small‐molecule screening assay, including the different methods available for mRNA isolation and analysis. Curr. Protoc. Chem. Biol . 4:49‐63 © 2012 by John Wiley & Sons, Inc.