REAL TIME PCR : An Introduction
Although the traditional methods of quantitating mRNA are fairly good such as northern blotting and in situ hybridization, they do not approach the ease and speed of Real Time PCR.
RT-PCR or reverse transcriptase PCR is semi-quantitative due to need to load samples on a gel and the insensitivity of ethidium bromide. Thus, real time PCR was developed out of the need to quantitate differences in mRNA expression in a easy and quick manner, and due to the need to use of small amounts of mRNA such as those obtained by small tissue samples, and LCM (laser capture microdissection) isolated cells.
Real-time reverse-transcriptase (RT) PCR is different from other quantitative PCR as it quantitates the initial amount of the template instead of detecting the amount of final amplified product (Freeman, 1999; Raeymaekers, 2000).
Real Time PCR is characterized by the point in time during cycling when amplification of the PCR product of interest is first detected rather than the amount of the PCR product of interest which is accumulated at the end-point after PCR which contained a large number of cycles. Real Time PCR does this by monitoring the amount of fluorescence emitted during the PCR reaction, and this acts as an indicator of the amount of PCR amplification that occurs during each PCR cycle. Thus, in newer Real Time PCR machines, one can visually see the progress of the reaction in "real time".
Real Time PCR also has a much wider dynamic range of up to 107-fold (compared to 1000-fold in conventional RT-PCR). The dynamic range of an assay determines how much the target concentration can vary and yet still be quantified. This wide dynamic range also results in a more accurate quantitation.