Real-Time qRT-PCR

The following techniques can be used to monitor degradation of the probe:

• intercalation of double-stranded DNA-binding dyes
• ³²P probe labeling
• labeling of the probe with fluorescent dyes

TaqMan assay (named after Taq DNA 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 Biosystems probes also include a minor groove binder for added specificity.

Applications of real time quantitative RT-PCR: relative and absolute quantification of gene expression, validation of DNA microarray results, variation analysis, counting bacterial, viral, or fungal loads, etc.

Nomenclature commonly used in real time quantitative RT-PCR:

Baseline is defined as PCR cycles in which a reporter fluorescent signal is accumulating but is beneath the limits of detection of the instrument.

ΔRn is an increment of fluorescent signal at each time point. The ΔRn values are plotted versus the cycle number.

Threshold 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. Threshold can be adjusted for each experiment so that it is in the region of exponential amplification across all plots.

Ct 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.