RTPCR

RTPCR, short for reverse transcription polymerase chain reaction, is a laboratory technique used to detect and analyze RNA. It combines reverse transcription, which converts RNA into complementary DNA (cDNA), with polymerase chain reaction (PCR) to amplify specific DNA targets. Conventional RTPCR is typically an endpoint method that yields a product detectable by gel electrophoresis or other post-amplification analysis, providing qualitative or semi-quantitative information about RNA presence.

In the standard workflow, RNA is isolated from cells or tissue and treated to remove genomic DNA. Reverse transcriptase enzymes synthesize cDNA from the RNA template, often using random primers, oligo-dT, or gene-specific primers. The resulting cDNA serves as the template for PCR amplification with primers specific to the target sequence. The amplified products are then detected after the reaction, usually by gel electrophoresis or sequencing.

Real-time RTPCR, also called RT-qPCR, monitors the accumulation of PCR products during the amplification process by measuring fluorescence. This enables quantitative analysis of RNA levels, often expressed as relative or absolute abundance. Detection methods include intercalating dyes such as SYBR Green and sequence-specific probes like TaqMan. RT-qPCR relies on careful control of reaction efficiency and appropriate normalization against reference genes.

Applications include gene expression profiling, diagnostic testing for RNA viruses, viral load monitoring, and research involving transcript variants or RNA quantification. Limitations involve RNA instability, potential DNA carryover, and the need for rigorous controls to avoid false positives or inaccurate quantification.