Reverse transcription polymerase chain reaction, abbreviated RT-PCR, detects mRNA, pre-mRNA and noncoding RNAs. RNA is converted to cDNA by reverse transcription and the product is amplified by PCR. A primer, which is a short nucleic acid sequence, is annealed to the complementary region on the RNA. The enzyme reverse transcriptase generates a single-stranded cDNA copy of a portion of the RNA. Specific synthetic DNA oligonucleotide primers or random hexamers are used. By using random hexamer priming cDNA copies of the entire length of mRNAs are possible. The cDNA is used as a template for PCR, where gene-specific primers are used to amplify a target region of DNA the corresponds to the RNA.
For analysis RT-PCR amplified products can be labelled and separated by gel electrophoresis. In real-time RT-PCR, also known as quantitative RT-PCR (RT-qPCR), data is collected while the reaction is occurring. Real-time RT-PCR measures the cycle threshold (Ct), the time at which fluorescence intensity is higher than background fluorescence. The greater the quantity of target DNA in the starting material, the less time it will take for a fluorescent signal to be detected. More starting material will yield a lower Ct. Detection chemistries for RT-PCR use light-emitting fluorescent fluorophores incorporated into DNA binding dyes, labelled hybridization probes, hydrolysis probes and hairpin probes.
