Reverse transcriptase-polymerase chain reaction RT-PCR

Reverse transcriptase-polymerase chain reaction (RT-PCR) Materials 

Moloney murine virus reverse transcriptase (superscript II GIB- 

Add 40 pi PCR buffer containing 50 pmol of the appropriate primers and 2 units taq polymerase. 

Amplify the template using standard PCR methods (35 rounds). 

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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). 

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. 

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... 

Eastwood SL, Burnet PW, Harrison PJ. 1997. GluR2 glutamate receptor subunit flip and flop isoforms are decreased in the hippocampal formation in schizophrenia A reverse transcriptase-polymerase chain reaction (RT-PCR) study. Brain Res Mol Brain Res 44 92-98. 

Direct immunohistochemical analysis of prostatic tissue has become very popular since the development of AR antibodies. However, a disadvantage of this technique in quantitative analysis is that the intensity of the immunohistochemical stain is dependent on the intactness of the structure of the AR. Therefore, mutations or alterations in the structure may reduce staining intensity (T5). Biochemical and immunohistochemical studies of AR content in relation to grade or stage of disease, as well as prediction of response to endocrine therapy, has been inconsistent. Nearly all primary prostate cancer specimens positively express AR protein, as determined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis as well as by immunohistochemical analysis on formalin-fixed, paraffin-embedded primary prostate tissues (D12, H14). In advanced-stage prostate cancer, immunohistochemical techniques has shown that metastases in bone, the... 

Three commercial assays for viral load determination are currently available Quantiplex (Chiron), NASBA (Organon-Teknika), and Amplicor (Roche). Only the latter would fall into the topic of this chapter (see Note 1), because it is based on the RNA- or reverse transcriptase-polymerase chain reaction (RT-PCR) technique. 

Enzyme-linked immunoabsorbent assays (ELISAs) for VHP infections can be performed on samples inactivated by treatment with P-propiolactone. Reverse transcriptase polymerase chain reaction (RT-PCR) tests on samples following RNA extraction using chloroform and methanol can detect most of the VHP agents rapidly. RT-PCR is particularly useful when isolation of the virus is difficult or impractical, and was effective in detecting the agent causing HPS months before it was isolated in culture (48). 

Jenkins, M.C., Trout, J., Abrahamsen, M.S., Higgins, J., and Fayer, R. 2000. Estimating viability of Cryptosporidium parvum oocysts using reverse transcriptase-polymerase chain reaction (RT-PCR) directed at mRNA encoding amyloglucosidase. J. Microbiol. Methods 34, 97-106. 

An introduction to single gene transcript quantification methods. A number of methods are now available for the detection of gene activity via messenger RNA (mRNA). In all protocols, the first step is to isolate total RNA. This can be probed directly or used for reverse transcriptase polymerase chain reaction (RT-PCR)... 

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