Methodological Approaches

Real time quantitative RT-PCR

The basis for mRNA quantitation in the ABI7700 (TaqMan) instrument is to continuously measure PCR product accumulation using a dual-labeled fluorogenic oligonucleotide probe, called a TaqMan™ probe. This probe is composed of a short (ca. 20-30 bases) oligodeoxynucleotide that is labeled with two different fluorescent dyes. On the 5' terminus is a reporter dye and on the 3' terminus is a quenching dye. This oligonucleotide probe sequence is homologous to an internal target sequence present in the PCR amplicon. When the probe is intact, energy transfer occurs between the two fluorophors and emission from the reporter is quenched by the quenching dye. During the extension phase of PCR, the probe is cleaved by 5' nuclease activity of Taq polymerase and thereby releasing the reporter from the oligonucleotide-quencher. This release from the quencher results in an increase in the reporter emission intensity. The ABI7700 uses fiber optic system that connects to each well in a 96-well PCR tray format. The laser light source excites each well and a CCD camera measures the fluorescence spectrum and intensity from each well to generate real-time data during PCR amplification. The ABI 7700 Prism software examines the fluorescence intensity of reporter and quencher dyes and calculates the increase in normalized reporter emission intensity over the course of the amplification. The results are then plotted versus time, represented by cycle number, to produce a continuous measure of PCR amplification.

Back to Methodological Approaches