PCR con transcriptase inversa
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A reacción en cadea da polimerase con transcritase inversa (ou PCR tras transcritase inversa), abreviada como RT-PCR (do inglés reverse transcriptase PCR) é unha das moitas variantes da reacción en cadea da polimerase (PCR) na que previamente se realiza unha reversotranscrición. This technique is commonly used in molecular biology to detect RNA expression.[1] RT-PCR is often confused with real-time polymerase chain reaction (qPCR) by students and scientists alike.[2] However, they are separate and distinct techniques. While RT-PCR is used to qualitatively detect gene expression through creation of complementary DNA (cDNA) transcripts from RNA, qPCR is used to quantitatively measure the amplification of DNA using fluorescent probes. qPCR is also referred to as quantitative PCR,[2] quantitative real-time PCR,[3] and real-time quantitative PCR.[4]
Although RT-PCR and the traditional PCR both produce multiple copies of particular DNA isolates through amplification, the applications of the two techniques are fundamentally different. Traditional PCR is used to exponentially amplify target DNA sequences. RT-PCR is used to clone expressed genes by reverse transcribing the RNA of interest into its DNA complement through the use of reverse transcriptase. Subsequently, the newly synthesized cDNA is amplified using traditional PCR.
In addition to the qualitative study of gene expression, quantitative PCR can be utilized for quantification of RNA, in both relative and absolute terms,[5] by incorporating qPCR into the technique. The combined technique, described as quantitative RT-PCR[6] or real-time RT-PCR[7] (sometimes even quantitative real-time RT-PCR[8]), is often abbreviated as qRT-PCR,[9] RT-qPCR,[10] or RRT-PCR.[11] Compared to other RNA quantification methods, such as northern blot, qRT-PCR is considered to be the most powerful, sensitive, and quantitative assay for the detection of RNA levels. It is frequently used in the expression analysis of single or multiple genes, and expression patterns for identifying infections and diseases.[5]
In order to avoid confusion, the following abbreviations will be used consistently throughout this article:
| Technique | Abbreviation |
|---|---|
| Polymerase chain reaction | PCR |
| Reverse transcription polymerase chain reaction | RT-PCR |
| Real-time polymerase chain reaction | qPCR |
| RT-PCR / qPCR combined technique | qRT-PCR |
Notas
- ↑ Freeman WM, Walker SJ, Vrana KE (January 1999). "Quantitative RT-PCR: pitfalls and potential". BioTechniques 26 (1): 112–22, 124–5. PMID 9894600.
- ↑ 2,0 2,1 Mackay, Ian (2007). Real-time PCR in Microbiology: From Diagnosis to Characterization. Norfolk, England: Caister Academic Press. p. 440. ISBN 1-904455-18-2.
- ↑ Radonić A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A (January 2004). "Guideline to reference gene selection for quantitative real-time PCR". Biochemical and Biophysical Research Communications 313 (4): 856–62. PMID 14706621. doi:10.1016/j.bbrc.2003.11.177. Consultado o 2012-11-06.
- ↑ Livak KJ, Schmittgen TD (December 2001). "Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method". Methods 25 (4): 402–8. PMID 11846609. doi:10.1006/meth.2001.1262. Consultado o 2012-11-06.
- ↑ 5,0 5,1 "groups.molbiosci.northwestern.edu" (PDF). Consultado o 2012-11-06.
- ↑ Joyce C (2002). "Quantitative RT-PCR. A review of current methodologies". Methods Mol. Biol. 193: 83–92. PMID 12325527. doi:10.1385/1-59259-283-X:083.
- ↑ Kang XP, Jiang T, Li YQ; et al. (2010). "A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus". Virol. J. 7: 113. PMC 2892456. PMID 20515509. doi:10.1186/1743-422X-7-113. Consultado o 2012-11-06.
- ↑ Bustin SA, Benes V, Nolan T, Pfaffl MW (June 2005). "Quantitative real-time RT-PCR--a perspective". J. Mol. Endocrinol. 34 (3): 597–601. PMID 15956331. doi:10.1677/jme.1.01755. Consultado o 2012-11-06.
- ↑ Varkonyi-Gasic E, Hellens RP (2010). "qRT-PCR of Small RNAs". Methods Mol. Biol. 631: 109–22. PMID 20204872. doi:10.1007/978-1-60761-646-7_10. Consultado o 2012-11-06.
- ↑ Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M (April 2010). "A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines". Methods 50 (4): S1–5. PMID 20215014. doi:10.1016/j.ymeth.2010.01.005. Consultado o 2012-11-06.
- ↑ Spackman E, Senne DA, Myers TJ; et al. (September 2002). "Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes". J. Clin. Microbiol. 40 (9): 3256–60. PMC 130722. PMID 12202562. doi:10.1128/jcm.40.9.3256-3260.2002. Consultado o 2012-11-06.