RTqPCR

RT-qPCR, or reverse transcription quantitative polymerase chain reaction, is a laboratory technique that combines reverse transcription of RNA into complementary DNA (cDNA) with real-time polymerase chain reaction to quantify RNA in a sample. It is widely used to measure gene expression and to detect RNA viruses, including clinical pathogens.

The typical workflow involves extracting RNA, converting it to cDNA by reverse transcription, and then amplifying the cDNA in real time using PCR. Fluorescence generated during amplification is monitored to quantify the target. Two common formats exist: one-step RT-qPCR, where reverse transcription and PCR occur in a single tube, and two-step RT-qPCR, where cDNA is synthesized first and then subjected to separate PCR. Fluorescence detection can employ DNA-binding dyes such as SYBR Green or sequence-specific probes like TaqMan. The cycle at which fluorescence crosses a defined threshold (the Ct or threshold cycle) is inversely related to the starting amount of target RNA. Absolute quantification uses a standard curve, while relative quantification compares expression to reference genes.

Interpreting results requires careful controls and normalization. RNA quality, inhibitors, and sample handling can influence outcomes. Contamination and non-specific amplification pose risks, particularly with dye-based detection. Appropriate controls (no-template controls, positive controls, and validated reference genes) are essential for reliable data.

Applications include clinical diagnostics for RNA viruses, gene expression profiling in research, pathogen surveillance, and validation of high-throughput data. RT-qPCR is valued for its sensitivity, speed, and quantitative capability, but accuracy depends on standardized protocols, proper controls, and rigorous data analysis.