Extensive detail on the application of the real‐time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high‐throughput, 384‐well‐format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real‐time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency‐corrected ΔCt method, and the comparative cycle time, or ΔΔCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT‐PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real‐time and non‐real‐time RT‐PCR applications.