Real-time reverse transcription polymerase chain reaction

Abstract

RT-qPCR is one of the most widespread and reliable methods of measuring gene expression. First, the extraction of mRNA from a homogeneous cell group is done in order to perform the RT phase (reverse transcription). At this point the reverse transcriptase enzyme synthesizes cDNA corresponding to each RNA strand. Then begins the qPCR (real time polymerase chain reaction), which gets its name because it gives quantitative results (“q” of qPCR), unlike the conventional PCR qualitative results. This stage is characterized by amplification of a specific site in the set of cDNA, which is achieved, briefly, through the use of a DNA-dependent thermostable DNA polymerase (Taq polymerase), two oligomers specific to serve as primers for polymerase and a non-specific DNA fluorophore (SYBR Green I, for example). As the double-strand cDNA is being synthesized, the fluorophore binds to its strands and, after being excited, emits light in proportion to the number of double chains. Through graphical analysis, it is possible to quantify the initial sample of cDNA that, in absence of errors, is proportional to mRNA