Gene Expression Detection Assay for Cancer Clinical Use

Narrandes, Shavira; Xu, Wayne

Many commercial companies provide qRT-PCR Taq polymerase enzyme and mixture reaction solutions. Besides the digital PCR that detects the actual copy of mRNAs and other alternative PCR methods, there are two major qRT-PCR systems: TaqMan and SYBR green. In TaqMan qRT-PCR, a specific probe labeled with a fluorescent reporter is bound on the cDNA of the target RNA. Taq polymerase enzyme will degrade the probes during the chain extension such that the fluorescent reporter is released. The amount of PCR product can be quantitatively measured by the fluorescence reporter detection. 10 As different reporter dyes can be utilized for a TaqMan assay, the sensitivity of a TaqMan assay is one of its major advantages. It also has a high specificity because of the specific probe but it cannot be used for multiplex gene targets. In SYBR Green qRT-PCR, the fluorescent dye SYBR Green binds with the amplified double stranded DNA and emits an intense green light. Therefore, the PCR products can be measured by detecting the bound SYBR Green fluorescence. Compared to TaqMan qRT-PCR, this method is inexpensive, easy to use, and can be used for multiplex gene targets. However, due to its ability to bind to any double-stranded DNA, nonspecific binding can lead to over quantification of PCR product. 9, 13 Data Analysis