Gene Expression Detection Assay for Cancer Clinical Use

Narrandes, Shavira; Xu, Wayne

Principle PCR (polymerase Chain Reaction) was first invented by Kary Mullis and his colleagues from the Cetus Corporation when they exploited the ability of Taq polymerase from Thermus Aquaticus, which amplifies DNA, to withstand elevated temperatures. In reverse transcription PCR (RT-PCR), RNA is first reverse transcribed into cDNA and then amplified. The first generation of RT-PCR uses a band's intensity in gel for a semi-quantification. 8 Real-time quantitative reverse transcription PCR (real-time qRT-PCR) and digital PCR were then developed. Instead of detecting how much PCR product is generated (conventional PCR), qRT-PCR counts how many reaction cycles are used to reach an amplification slope status in real-time. The Ct (cycle threshold) levels, defined as the number of cycles required for the fluorescent signal to cross the threshold (i.e. exceeds background level) are inversely proportional to the amount of target nucleic acid in the sample (i.e. the lower the Ct level, the greater the amount of target nucleic acid in the sample). The first step requires reverse transcription of the mRNA template. Then, a primer pair anneals to the target cDNA template and a DNA polymerase (e.g. Taq) extends from the ligated primers. A thermal cycler varies the temperature settings to perform the three stages of the PCR: denaturation, annealing, and extension. Finally, a standard dissociation curve can be studied to determine the initial amount of RNA (cDNA) transcripts. 9, 10 Primer design