Quantitative Real-Time PCR (qPCR) uses fluorescent reporter molecules to allow quantification of amplified products. As in conventional PCR, a DNA, cDNA, or RNA template is amplified, but at each cycle, fluorescent signals are monitored for relative or absolute quantification. This technique is useful for numerous areas of research including gene expression analysis, genotyping, microRNA analysis, genetic variation analysis, and protein analysis.
The fluorescence of the reporter molecules is measured and quantified; typically these are either dyes (i.e. SYBR® Green, Ethidium Bromide) that intercalate between the bases in double-stranded DNA, or probes designed to bind a specific sequence (i.e. Molecular Beacons, TaqMan® probes) on the DNA.
There are two major quantification methods for qPCR data. Relative quantification, the more common method, uses ΔΔCt information, by which the expression or abundance ratio of the target gene in the sample is determined, compared to a control gene, and normalized with the expression ratio of a reference gene. Since, the efficiency E values for target and reference genes differ, this method also accounts for the differences in E values. This method is more frequently employed in gene expression analysis.
The second method, absolute quantification, is more common in environmental microbiology and makes use of the standard-curve (SC). The SC method uses dilution series of known template concentration, N0, to create a standard curve using the linear regression of log(N0) versus the CT (threshold cycle). This is then used to calculate template concentrations of the sample. The basis of this method is that the efficiency value of the sample is the same as that of the standard.