Reverse transcriptase/polymerase chain reaction

Reverse transcriptase polymerase chain reaction (RT-PCR) is a molecular reaction used for the amplification and detection of RNA (mRNA) after transcription in to cDNA. 

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This acronym stands for Reverse Transcriptase Polymerase Chain Reaction, a method used to first copy a strand of RNA into cDNA, then amplify it through standard PCR methods. 

RT-PCR reverse transcriptase polymerase chain reaction RTV ritonavir... 

Dunne, A. L., and Crowe, S. M. (1997). Comparison of branched DNA and reverse transcriptase polymerase chain reaction for quantifying six different HTV-1 subtypes in plasma. AIDS 11,2-3. 

Abrahamsen HN, Steiniche T, Nexo E, et al. Towards quantitative mRNA analysis in paraffin-embedded tissues using real-time reverse transcriptase-polymerase chain reaction. A methodological study on lymph nodes from melanoma patients. J. Mol. Diagn. 2003 5 34-41. 

Cronin M, Pho M, Dutta D, et al. Measurement of gene expression in archival paraffin-embedded tissues development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay. Am. J. Pathol. 2004 164 35—42. 

Mangham DC, Williams A, McMullan DJ, et al. Ewing s sarcoma of bone the detection of specific transcripts in a large, consecutive series of formalin-fixed, decalcified, paraffin-embedded tissue samples using the reverse transcriptase -polymerase chain reaction. Histopathology 2006 48 363-376. 

The RNA molecules, ribosomal RNA (rRNA) and messenger RNA (mRNA) play key roles in the protein synthesis. The amount of RNA in individual cells or in a community may, therefore, be taken as an indicator of protein synthesis and, thus, microbial activity. The number of active cells can be detected by fluorescent in situ hybridization (FISH) (Amann et al. 1995). By this method, individual cells carrying high concentrations of rRNA, situated on ribosomes, are quantified by fluorescence microscopy. The amount of rRNA in a community can also be detected by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), where rRNA extracted from soil is detected by creating a DNA copy and separating by gel electrophoresis (Duineveld et al. 2001). 

List of Abbreviations cDNA, complementary DNA ddH20, double-distilled H2O dNTP, deoxyribonu-cleotide triphosphate EDTA, ethylenediaminetetraacetic acid MgCl2, magnesium chloride mRNA, messenger ribonucleic acid NaOH, sodium hydroxide PCR, polymerase chain reaction qRT PCR, quantitative reverse transcriptase polymerase chain reaction RNase, ribonuclease RT PCR, reverse transcriptase polymerase chain reaction UTR, untranslated region... 

Abbreviation Bp, nucleotide base pairs cDNA, complementary DNA ChIP, chromatin Immunoprecipi-tation Cy5, cyanine 5-dCTP Cy3, cyanine 3-dCTP ESTs, expressed sequence tags FDR, false discovery rate MIAME, minimum information about a microarray experiment mRNA, RNA, messenger NIA, National Institutes of Aging RFUs, relative fluorescence units RT-PCR, reverse transcriptase polymerase chain reaction SAGE, serial analysis of gene expression SAM, significance analysis of microarrays... 

Regarding reverse transcriptase polymerase chain reaction (RT-PCR) analysis to assess a splice site variant, as seen in Fig. 12.6, if one designs forward (F-) and reverse (R-) RT-PCR primers to span the SNP (which in turn creates or abolishes a splice site), one wiU have different size PCR products (labeled a, b, and c in the figure) that can easily be resolved on a gel. 

Chu, K. H., Wong, S. H., and Leung, P. S. C. (2000). Tropomyosin is the major mollusk allergen Reverse transcriptase polymerase chain reaction, expression and IgE reactivity. Mar. Biotechnol. 2,499-509. 

E. Therapeutic response Efficacy of Infergen therapy was determined by measurement of serum alanine aminotransferase (ALT) concentrations at the end of therapy (24 weeks) and following 24 weeks of observation after the end of treatment of adults with chronic HCV infection. Serum HCV RNA was also assessed using a quantitative reverse transcriptase polymerase chain reaction (RT-PCR). At the end of 24 weeks of treatment, ALT normalization was observed in 39% of patients on Infergen and in 35% of patients on interferon alfa-2b Intron A). Only 17% of patients in each group... 

Galvan, B. Christopoulos, T. K. Quantitative reverse transcriptase-polymerase chain reaction for prostate-specific antigen mRNA. Clin. Biochem. 1997, 30(5), 391-397. 

Heniford, B W., Shum-Siu, A, Leonberger, M., and Hendler, F J (1993) Variation m cellular EGF receptor mRNA expression demonstrated by in situ reverse transcriptase polymerase chain reaction. Nucleic Acids Res 21,3159-3166... 

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

The methods used for the evaluation of regulation of gene expression are too numerous to be described in detail here. They include Northern analysis to determine levels of a particular mRNA, nuclear run on to determine whether an increase in mRNA is due to an increase in the rate of transcription, and promoter deletion analysis to identify specific elements in the promoter region responsible for the control of expression. Of much current interest is the use of microarrays that permit the study of the expression of hundreds to thousands of genes at the same time. Reverse transcriptase-polymerase chain reaction and RNase protection assay techniques are used to amplify and quantitate mRNAs, while the electrophoretic mobility shift assay is used to measure binding of a transcription factor to its specific DNA consensus sequence. 

Fig. 2. Expression map of human tissue kallikreins in a variety of tissues, as determined by reverse transcriptase polymerase chain reaction. The relative semiquantitative expression levels for each gene are indicated.

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