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Reverse transcription polymerase chain reaction

Reverse transcription polymerase chain reaction

Method in molecular biology; variant of polymerase chain reaction (pcr), is a technique commonly used in molecular biology to detect rna expression

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.

Timeline

Further Resources

Title
Author
Link
Type
Date

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

Journal

June 4, 2021

Homologous Recombination Deficiency (HRD) Testing in Advanced Ovarian Cancer

Web

September 29, 2020

Reverse Transcription Polymerase Chain Reaction - an overview | ScienceDirect Topics

Web

News

Title
Author
Date
Publisher
Description
BS Web Team & Agencies
June 6, 2020
@bsindia
The test kit has been developed at a cost of Rs 550 and it can be reduced to up to Rs 350 when taken to mass production

References

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