Real‐time PCR is a recent modification to the polymerase chain reaction that allows precise quantification of specific nucleic acids in a complex mixture by fluorescent detection of labeled PCR products. Detection can be accomplished using specific as well as nonspecific fluorescent probes. Real‐time PCR is often used in the quantification of gene expression levels. Prior to using real‐time PCR to quantify a target message, care must be taken to optimize the RNA isolation, primer design, and PCR reaction conditions so that accurate and reliable measurements can be made. This short overview of real‐time PCR discusses basic principles behind real‐time PCR, some optimization and experimental design considerations, and how to quantify the data generated using both relative and absolute quantification approaches. Useful Web sites and texts that expand upon topics discussed are also listed. Curr. Protoc. Essential Lab. Tech. 8:10.3.1‐10.3.40. © 2014 by John Wiley & Sons, Inc.

Critical to the success of real-time RT-PCR for measuring gene expression levels was the development of procedures for converting an RNA population into a DNA copy (cDNA). Retroviral-derived, RNA-dependent, DNA polymerases (reverse transcriptases) were the first polymerases shown to be Current Protocols Essential Laboratory Techniques 10.