Analysis of variant gene family expression by quantitative PCR

Real-time polymerase chain reaction (PCR), or quantitative PCR (qPCR), is a rapid, sensitive, and specific method used for a broad variety of applications including quantitative gene expression analysis, DNA copy number measurement, characterization of gene and chromosomal deletions, and genotyping. Real-time reverse transcription (RT)-PCR has largely supplanted Northern blot and RNase protection assays, as two examples, as a means of quantifying transcript levels. The method utilizes small amounts of RNA and allows efficient screening of a large number of samples. Here, we describe the materials and methods required to perform real-time RT-PCR, including RNA purification, cDNA synthesis, and real-time PCR analysis of cDNA samples.