Quantitative PCR (qPCR), also known as real-time PCR, is a laboratory technique that amplifies and simultaneously quantifies DNA in real time. Unlike conventional PCR, which only shows the final amount of DNA, qPCR monitors the accumulation of DNA throughout the reaction using fluorescence.
How qPCR Works
- Reaction Setup
The reaction contains the same components as conventional PCR—template DNA, primers, DNA polymerase, and nucleotides—plus a fluorescent reporter. Two common reporter systems are SYBR Green, a dye that fluoresces when bound to double-stranded DNA, and TaqMan probes, which are sequence-specific fluorescent probes.
- Amplification and Detection
The reaction undergoes the same thermal cycling as standard PCR: denaturation, annealing, and extension. After each cycle, a fluorescence measurement is taken. As more DNA is amplified, the fluorescence signal increases proportionally.
- The Ct Value
The threshold cycle (Ct) is the cycle number at which the fluorescence signal rises above the background level. The Ct value is inversely proportional to the starting amount of target DNA: more starting DNA means fewer cycles are needed to reach the threshold.
- Quantification
Absolute quantification uses a standard curve of known DNA concentrations to calculate the exact amount of target DNA. Relative quantification compares the Ct values of a target gene to a reference gene, determining fold changes in expression.
- Melt Curve Analysis
When SYBR Green is used, a melt curve analysis is performed after amplification. The DNA is slowly heated while fluorescence is monitored. Each DNA product melts at a characteristic temperature, confirming that the correct amplicon was produced and no primer-dimers are present.
