What is RT-qPCR and what is it used for?

• A. End-point PCR to detect presence of DNA
• B. Real-time quantitative PCR starting from RNA to quantify gene expression levels
• C. Protein assay using quantitative colorimetric readout
• D. DNA sequencing method for mutation detection

Answer: (B)

RT-qPCR measures RNA by converting it to DNA and then amplifying that DNA while measuring fluorescence in real time. This allows you to quantify how much of a specific RNA transcript is present, which is why it’s ideal for assessing gene expression levels. The process starts with RNA and uses reverse transcriptase to make cDNA, followed by quantitative PCR where the amount of product is tracked cycle by cycle. The resulting Ct values reflect the starting RNA abundance, and expression levels are typically compared across samples after normalizing to stable reference genes. This real-time, RNA-based quantification distinguishes RT-qPCR from traditional end-point PCR (which only asks if a product is present), from protein assays, or from sequencing-based mutation detection. In practice, you extract RNA, reverse-transcribe it, and perform qPCR with gene-specific primers and a fluorescent reporter, enabling sensitive and dynamic measurement of gene expression.