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A technique used in molecular biology, real-time PCR is an application of PCR technology used to determine the levels of a specific RNA (usually) in a sample. Actually it's real-time reverse transcriptase PCR, which would be RTRTPCR.

As implemented by most machine makers, real-time PCR requires a polymerase with 5'-exonuclease activity. The reaction involves the template in the sample, two primers separated by less than 100 bp, and a special probe corresponding to the middle, amplified sequence. The probe is labeled with both a fluorescent tag and a quencher molecule. The PCR reaction occurs in special tubes and in a special thermocycler with a lamp and fluorescent detector connected to the reaction tubes with fiber optics. As the PCR reaction takes place, the 5'-exonuclease activity cleaves the probe, separating the fluorescent tag from the quencher. This results in increased fluorescence as detected by the machine.

As this reaction involves three independent hybridizations, background is low and false signals are extremely rare. Using known quantities of template, real-time PCR can tell you how many copies of an mRNA is present in a sample, in a far more precise manner than other techniques.

This technique may eventually replace the Northern Blot, though currently the price of the machine and the specially made probes make the procedure expensive.

There are other ways of doing real-time PCR that simply use dyes that bind double-stranded DNA. These techniques are cheaper, but not as robust or precise as the described method.

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