Real-time reverse-transcriptase

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. 

Rokbi, B., Seguin, D., Guy, B., Mazarin, V., Vidor, E., Mion, F., Cadoz, M., and Quentin-Millet, M.-J. (2001). Assessment of Helicobacter pylori gene expression within mouse and human gastric mucosae by real-time reverse transcriptase PCR. Infect. Immun. 69, 4759 766. 

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. 

Endrizzi K, Fischer J, Klein K, Schwab M, Nussler A, Neuhaus P, Eichelbaum M, Zanger UM. Discriminative quantification of cytochrom P4502D6 and 2D7/8 psuedogene expression by TaqMan real-time reverse transcriptase polymerase chain reaction. Anal Biochem 2002 15 121-131. 

Figure 11 TaqMan RT-PCR assay. Abbreviation RT-PCR, real-time reverse transcriptase-polymerase chain reaction.

The authors would like to thank Dr. Thomas Rushmore, Dr. Karen Richards, and Ms. Kristie Strong-Basalyga for their contribution in the development of the quantitative real-time reverse transcriptase-polymerase chain reaction assay for CYP analysis in primary hepatocytes. 

Bowen WP, Carey J, Miah A, et al. Measurement of cytochrome P450 gene induction in human hepatocytes using quantitative real-time reverse transcriptase-polymerase chain reaction. Dmg Metab Dispos 2000 28 781-788. 

Palmer S, Wiegand AP, Maldarelli F, Bazmi H, Mican JM, PoUs M, Dewar RL, Planta A, Liu S, Metcalf JA, Mellors JW, Coffin JM. New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J. Clin. Microbiol. 2003 41 4531-4536. 

Bijwaard, K.E., Fetsch, J.F., Przygodzki, R. et al. (2002) Detection of SYT-SSX fusion transcript in archival synovial sarcoma by Real-time reverse-transcriptase polymerase chain reaction. J Mol Diag, 4, 59-64. 

Kurahashi, T., Hara, L, Oka, N. et al. (2005) Detection of micrometastases in pelvic lymph nodes in patients undergoing radical cystectomy for focally invasive bladder cancer by real-time reverse transcriptase-PCR for cytokeratinl9 and uroplakin II. Clin Cancer Res, 11, 3773-3777. 

Vanlandingham, D.L., Schneider, B.S., Klingler, K., Fair, J., Beasley, D., Huang, J., Hamilton, P. and Higgs, S. (2004) Real-time reverse transcriptase-polymerase chain reaction quantification of West Nile virus transmitted by Culex pipiens quinquefasciatus. Am. J. Prop. Med. Hyg. 71, 120-123. 

Johnson, B. W. Russell, B. J. Lanciotti, R. S. Serotype-specific detection of dengue viruses in a fourplex real-time reverse transcriptase PCR assay. J. Clin. Microbiol. 2005, 43,4977-4983. 

Cook, R. F Cook, S. J. Li, K Montelaro, R. C. Issel, C. J. Development of a multiplex real-time reverse transcriptase-polymerase chain reaction for equine infectious anemia virus (EIAV). J. Virol. Methods 2002,105, 171-179. 

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