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.
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Further Resources
Comparison of Reverse Transcription-Polymerase Chain Reaction, Immunohistochemistry, and Fluorescence In Situ Hybridization Methodologies for Detection of Echinoderm Microtubule-Associated Proteinlike 4-Anaplastic Lymphoma Kinase Fusion-Positive Non-Small Cell Lung Carcinoma: Implications for Optimal Clinical Testing
Wallander, Michelle L., Geiersbach, Katherine B., Tripp, Sheryl R., Layfield, Lester J.
Web
July 1, 2012
Development of an efficient one-step real-time reverse transcription polymerase chain reaction method for severe acute respiratory syndrome-coronavirus-2 detection
Yukiko Nakura, Heng Ning Wu, Yuya Okamoto, Muneyuki Takeuchi, Koichiro Suzuki, Yoshitaka Tamura, Yuichiro Oba, Fumiko Nishiumi, Nobuaki Hatori, Shinsuke Fujiwara, Kiyoshi Yasukawa, Shinobu Ida, Itaru Yanagihara
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June 4, 2021
Homologous Recombination Deficiency (HRD) Testing in Advanced Ovarian Cancer
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September 29, 2020
Reverse transcription polymerase chain reaction
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Reverse Transcription Polymerase Chain Reaction - an overview | ScienceDirect Topics
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