Quantitative TaqMan Real-Time PCR: Diagnostic and Scientific Applications

The invention of real-time polymerase chair reaction (PCR) has revolutionized the quantification of gene expression and DNA copy number measurements. However, after the first documentation of real-time PCR in 1993 (1 ), it took several years for this method to become a mainstream tool. PCR generates DNA copies in an exponential way. As soon as resources are exhausted, however, the so-called plateau phase of PCR reaction is reached, making quantification very unreliable. Therefore, quantification appears most reliable in the early exponential phase of PCR (i.e., in a “real-time” fashion). To ensure measurements in this phase of the PCR cycle, real-time PCR measures as soon as the threshold of detection is definitely reached. The cycle of PCR at which this occurs is then named the threshold cycle (2 ) (see Fig. 1 ). It is the objective of this chapter to describe the possibilities of TaqMan real-time PCR for mRNA and DNA quantification and to discuss pitfalls and alternatives.