qPCR

Quantitative PCR (qPCR), also known as real-time PCR, is a laboratory technique that amplifies DNA and measures the amount of DNA produced in real time during each cycle. It can quantify target DNA in a sample or measure gene expression by quantifying reverse-transcribed RNA (RT-qPCR).

Principle: The technique relies on the exponential amplification of a specific DNA region. Fluorescence is generated by DNA-binding dyes or sequence-specific probes and increases proportionally with product accumulation, allowing monitoring in real time. The cycle threshold (Ct) is the number of cycles required for the fluorescence to cross a predefined threshold; lower Ct indicates higher starting template.

Procedure: DNA or RNA is isolated, RNA may be reverse transcribed to cDNA, and a reaction mix containing primers, a DNA polymerase, nucleotides, buffer, and a fluorescent reporter is prepared. The sample is cycled in a real-time PCR instrument that records fluorescence after each cycle. Melt-curve analysis or probe specificity can verify product specificity.

Data analysis: Quantification can be absolute, using a standard curve of known template amounts, or relative, using reference genes for normalization and the ΔΔCt method to compare samples. Consistency of amplification efficiency is important for accurate results.

Applications and considerations: qPCR is widely used for gene expression analysis, pathogen quantification, copy-number variation studies, and diagnostic assays. Important considerations include primer design and amplicon length, choice of reporter chemistry, controls for inhibition and contamination, and validation of reference genes.