Real-Time qRT-PCR (Real-Time Quantitative Reverse Transcription PCR) is a major development of PCR technology that enables reliable detection and measurement of products generated during each cycle of the PCR process. This technique became possible after the introduction of an oligonucleotide probe which was designed to hybridize within the target sequence. Cleavage of the probe during PCR because of the 5′ nuclease activity of Taq polymerase can be used to detect amplification of the target-specific product.
assay (named afterTaqDNA polymerase) was one of the earliest methods introduced for real-time PCR reaction monitoring and has been widely adopted for both the quantification of mRNAs and for detecting variation. The method exploits the 5′ endonuclease activity of Taq DNA polymerase to cleave an oligonucleotide probe during PCR, thereby generating a detectable signal. The probes are fluorescently labeled at their 5′ end and are non-extendable at their 3′ end by chemical modification. Specificity is conferred at three levels: via two PCR primers and the probe. Applied Biosystemsprobes also include a minor groove binder for added specificity.
is defined as PCR cycles in which a reporter fluorescent signal is accumulating but is beneath the limits of detection of the instrument.
is an increment of fluorescent signal at each time point. The ΔRn values are plotted versus the cycle number.
is an arbitrary level of fluorescence chosen on the basis of the baseline variability. A signal that is detected above the threshold is considered a real signal that can be used to define the threshold cycle (Ct) for a sample. The threshold can be adjusted for each experiment so that it is in the region of exponential amplification across all plots.
is defined as the fractional PCR cycle number at which the reporter fluorescence is greater than the threshold. The Ct is a basic principle of real-time PCR and is an essential component in producing accurate and reproducible data.
Article Origin: National Libary of Medicine
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