qPCR
Quantitative Real-Time PCR (qPCR) is an advanced PCR technique that utilizes fluorescent reporter molecules to monitor the amplification of DNA, cDNA, or RNA in real-time, enabling the quantification of amplified products during each cycle. Unlike conventional PCR, where only the presence or absence of the target is detected, qPCR provides quantitative data through the measurement of fluorescent signals, allowing for both relative and absolute quantification of the target. This powerful technique is widely used in various fields of research, including gene expression analysis, genotyping, microRNA analysis, genetic variation studies, and protein analysis, providing crucial insights into the regulation and function of genes and proteins.
Overview
How to Analyze qPCR Data
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.
Relative Quantitation of qPCR Data
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.
Absolute Quantitation of qPCR Data
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.