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Reverse transcription polymerase chain reaction
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== Application == The exponential amplification via reverse transcription polymerase chain reaction provides for a highly sensitive technique in which a very low copy number of RNA molecules can be detected. RT-PCR is widely used in the diagnosis of genetic diseases and, semiquantitatively, in the determination of the abundance of specific different RNA molecules within a cell or tissue as a measure of [[Gene expression#Techniques and tools|gene expression]]. === Research methods === RT-PCR is commonly used in research methods to measure gene expression. For example, Lin et al. used qRT-PCR to measure expression of Gal genes in yeast cells. First, Lin et al. engineered a mutation of a protein suspected to participate in the regulation of Gal genes. This mutation was hypothesized to selectively abolish Gal expression. To confirm this, gene expression levels of yeast cells containing this mutation were analyzed using qRT-PCR. The researchers were able to conclusively determine that the mutation of this regulatory protein reduced Gal expression.<ref name="pmid22308403">{{cite journal |vauthors=Lin L, Chamberlain L, Zhu LJ, Green MR | title = Analysis of Gal4-directed transcription activation using Tra1 mutants selectively defective for interaction with Gal4 | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 109 | issue = 6 | pages = 1997–2002 |date=February 2012 | pmid = 22308403 | pmc = 3277556 | doi = 10.1073/pnas.1116340109 | bibcode = 2012PNAS..109.1997L | doi-access = free }}</ref> [[Northern blot]] analysis is used to study the RNA's gene expression further. === Gene insertion === RT-PCR can also be very useful in the insertion of [[eukaryotic]] genes into [[prokaryotes]]. Because most eukaryotic genes contain [[introns]], which are present in the genome but not in the mature mRNA, the cDNA generated from a RT-PCR reaction is the exact (without regard to the error-prone nature of reverse transcriptases) DNA sequence that would be directly translated into [[protein]] after [[transcription (biology)|transcription]]. When these genes are expressed in prokaryotic cells for the sake of protein production or purification, the RNA produced directly from transcription need not undergo splicing as the transcript contains only [[exons]]. (Prokaryotes, such as E. coli, lack the mRNA splicing mechanism of eukaryotes). === Genetic disease diagnosis === RT-PCR can be used to diagnose [[genetic disease]] such as [[Lesch–Nyhan syndrome]]. This genetic disease is caused by a malfunction in the [[HPRT1]] gene, which clinically leads to the fatal [[urinary stone|uric acid urinary stone]] and symptoms similar to [[gout]].{{clarify|text=[6]|date=April 2020}} Analyzing a pregnant mother and a [[fetus]] for mRNA expression levels of HPRT1 will reveal if the mother is a carrier and if the fetus will likely to develop Lesch–Nyhan syndrome.<ref name="pmid23046577">{{cite journal |vauthors=Torres RJ, Garcia MG, Puig JG | title = Carrier and prenatal diagnosis of Lesch-Nyhan disease due to a defect in HPRT gene expression regulation | journal = Gene | volume = 511 | issue = 2 | pages = 306–7 |date=December 2012 | pmid = 23046577 | doi = 10.1016/j.gene.2012.09.121 }}</ref> === Cancer detection === Scientists are working on ways to use RT-PCR in [[cancer]] detection to help improve [[prognosis]], and monitor response to therapy. Circulating [[tumor cell]]s produce unique mRNA transcripts depending on the type of cancer. The goal is to determine which mRNA transcripts serve as the best [[biomarkers]] for a particular cancer cell type and then analyze its expression levels with RT-PCR.<ref name="pmid17525108">{{cite journal |vauthors=Xi L, Nicastri DG, El-Hefnawy T, Hughes SJ, Luketich JD, Godfrey TE | title = Optimal markers for real-time quantitative reverse transcription PCR detection of circulating tumor cells from melanoma, breast, colon, esophageal, head and neck, and lung cancers | journal = Clin. Chem. | volume = 53 | issue = 7 | pages = 1206–15 |date=July 2007 | pmid = 17525108 | doi = 10.1373/clinchem.2006.081828 | doi-access = free }}</ref> RT-PCR is commonly used in studying the genomes of [[virus]]es whose genomes are composed of RNA, such as [[Influenzavirus A]], [[retrovirus]]es like [[HIV]] and [[SARS-CoV-2]].<ref>{{cite web |title=Coronavirus: il viaggio dei test |url=https://www.iss.it/web/guest/primo-piano/-/asset_publisher/o4oGR9qmvUz9/content/id/5269706 |website=Istituto Superiore di Sanità}}</ref> === Pathogen detection === [[Polymerase chain reaction#Infectious disease applications|PCR tests]] can be used for early detection of DNA-based pathogens through the amplification of pathogenic DNA, even before the host begins producing [[antibodies]].<ref name="MedlinePlus 24">{{cite web |last1=Medline Plus |title=PCR Tests |url=https://medlineplus.gov/lab-tests/pcr-tests/ |website=National Library of Medicine |publisher=National Institutes of Health |access-date=21 March 2025 |archive-url=https://web.archive.org/web/20250316060703/https://medlineplus.gov/lab-tests/pcr-tests/ |archive-date=16 March 2025 |date=19 September 2024 |url-status=live}}</ref> RT-PCR allows this process to be extended to RNA-based pathogens through the amplification of cDNA reverse-transcribed from a given pathogen's RNA.<ref name="iaea/covid_rt-rt-pcr"></ref> RT-PCR tests are best known for their use in [[COVID-19 testing]]<ref name="Cleveland Clinic 25">{{cite web |last1=Cleveland Clinic |title=PCR Test |url=https://my.clevelandclinic.org/health/diagnostics/21462-covid-19-and-pcr-testing |access-date=21 March 2025 |archive-url=https://web.archive.org/web/20250304035130/https://my.clevelandclinic.org/health/diagnostics/21462-covid-19-and-pcr-testing |archive-date=4 March 2025 |date=21 January 2025 |url-status=live}}</ref> but have also been used to diagnose diseases such as [[Ebola]],<ref name="iaea/covid_rt-rt-pcr"></ref> [[Zika]],<ref name="iaea/covid_rt-rt-pcr"></ref> [[MERS]],<ref name="iaea/covid_rt-rt-pcr"></ref> [[SARS]]<ref name="iaea/covid_rt-rt-pcr"></ref> and [[influenza]].<ref name="Cleveland Clinic 25></ref>
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