Quantitative Reverse Transcriptase Polymerase Chain Reaction

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Abstract

Since the first documentation of real-time polymerase chain reaction (PCR),1 it has been used for an increasing and diverse number of applications, including mRNA expression studies, DNA copy number measurements in genomic or viral DNAs,2–7 allelic discrimination assays,8,9 expression analysis of specific splice variants of genes10–13 and gene expression in paraffin-embedded tissues,14,15 and laser captured microdissected cells.13,16–19 Therefore, quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) is now essential in molecular diagnostics to quantitatively assess the level of RNA or DNA in a given specimen. QRT-PCR enables the detection and quantification of very small amounts of DNA, cDNA, or RNA, even down to a single copy. It is based on the detection of fluorescence produced by reporter probes, which varies with reaction cycle number. Only during the exponential phase of the conventional PCR reaction is it possible to extrapolate back in order to determine the quantity of initial template sequence. The “real-time” nature of this technology pertains to the constant monitoring of fluorescence from specially designed reporter probes during each cycle. Due to inhibitors of the polymerase reaction found with the template, reagent limitation or accumulation of pyrophosphate molecules, the PCR reaction eventually ceases to generate template at an exponential rate (i.e., the plateau phase), making the end point quantitation of PCR products unreliable in all but the exponential phase.