RealTimePCR

RealTimePCR, commonly referred to as real-time quantitative PCR or qPCR, is a molecular biology method that monitors DNA amplification during each cycle of the polymerase chain reaction. By measuring fluorescence emitted by reporter molecules, it allows quantification of the initial amount of target nucleic acid in a sample in real time, contrasting with end-point PCR.

The fluorescence arises from DNA-binding dyes such as SYBR Green or from sequence-specific probes like TaqMan. As amplification proceeds, signal increases in proportion to product yield. The cycle threshold (Ct) value, at which fluorescence exceeds a set threshold, is used to estimate starting copy numbers, often via standard curves or comparative methods.

Applications include gene expression analysis by RT-qPCR (converting RNA to cDNA before amplification), pathogen detection, DNA copy-number assessment, genotyping, and validation of sequencing results. Real-time measurement provides a faster, more sensitive, and quantitative alternative to conventional PCR.

Considerations include assay design, primer/probe specificity, and amplification efficiency near 100%. Normalization to stable reference genes is common in RT-qPCR. Data analysis requires controls for inhibition and contamination, and interpretation of results depends on proper experimental design and statistical methods.