Real Time PCR  Real time PCR was developed as a means to quantitate differences in mRNA expression. PCR methods are particularly valuable when amounts of RNA are low since the fact that they involve an amplification step means they are more sensitive.
Real Time PCR
Real Time PCR is used to determine the quantity of DNA real-time and thus a subset of the Q-PCR methods. It uses fluorescent dyes or fluorophore-DNA probes to measure the amount of amplification in real time. This is used to infer starting amount. Real-time PCR is often confusingly abbreviated as RT PCR, which overlaps with reverse transcription PCR; QRT-PCR or RTQ-PCR are better acronyms. In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (QRT-PCR) or kinetic polymerase chain reaction, is a laboratory technique used to simultaneously quantify and amplify a specific part of a given DNA molecule. It is used to determine whether or not a specific sequence is present in the sample; and if it is present, the number of copies in the sample. It is the real-time version of quantitative polymerase chain reaction (Q-PCR), itself a modification of polymerase chain reaction. The procedure follows the general pattern of polymerase chain reaction, but the DNA is quantified after each round of amplification; this is the "real-time" aspect of it. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-strand DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA. Frequently, real-time polymerase chain reaction is combined with reverse transcription polymerase chain reaction to quantify low abundance messenger RNA (mRNA), enabling a researcher to quantify relative gene expression at a particular time, or in a particular cell or tissue type. Although real-time quantitative polymerase chain reaction is often marketed as RT-PCR, it should not to be confused with reverse transcription polymerase chain reaction, also known as RT-PCR.
Real time PCR is the most sensitive and reproducible method for quantifying DNA target concentration in biological solutions by means of the polymerase chain reaction. More sensitive, because assays are designed to function at maximal efficiency over greater than 5 orders of magnitude, and more reproducible because quantification data is collected early in the exponential phase of the PCR where variability is reduced over that seen using 'end point' readings (Fig.1). Although there are a number of different systems for quantifying PCR reactions in real time, they have in common, the emission of amplification dependent florescence. Flurescence production (Y axis) is measured during each PCR cycle (x-axis) to give an 'amplification plot'. The cycle at which fluorescence release exceeds the background level by 10 fold is set as the Threshold (CT value). The cycle at which this occurs is dependent on the starting concentration of the target template, and quantification of this data is taken at this point. Simply adding an intercalating agent to the reaction, that fluoresces when bound to double stranded DNA, will produce amplification dependent fluorescence (e.g SYBR green). This has the advantage of being cheap and sensitive, but will detect all double stranded PCR products i.e primer dimers and mis-primed products. For this reason a system of fluorescent dyes with quenchers is often preferable. Prior to amplification, the fluorescent dye and quencher remain in close proximity and the quencher prevents fluorescence emission. During amplification the dye and quencher are physically separated resulting in 'de-quenching' and the fluorescence emission. Taqman ä probes are one of many ways of organising the dye and quencher, and work by sighting a DNA probe between the forward and reverse primers that has the fluorescent dye at the 5' end and a quencher at the 3' end (Fig.2). During amplification, the polymerase cleaves the probe, separating the dye and quencher. Since all three primers must bind correctly to produce an amplification plot, signals are highly unlikely to result from mis-priming events, additionally, the probe will not be cleaved during primer dimer formation.
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