Abstract
Reverse-transcriptase-polymerase chain reaction has become one of the most widely applied techniques in biomedical research. The ease with which the technique permits specific mRNA to be detected and quantified has been a major asset in the molecular investigation of disease pathogenesis. Disease-related imbalances in the expression of specific mRNAs can be sensitively and quantitatively determined by RT-PCR. RT-PCR also offers many opportunities in diagnostics, allowing sensitive detection of RNA viruses such as Human Immunodeficiency Virus (HIV)and Hepatitis C Virus (HCV). RT-PCR is an integral component of many methodologies that are essential to biomedical research, including in situ localization of mRNA, antibody engineering, and cDNA cloning. This chapter provides an overview of some of the ways in which RT-PCR can be utilized in biomedical science, and summarizes the importance and applicability of the protocols described in this volume. These protocols include some useful adaptations and applications that may have significance for those in the wider research community who are already familiar with the basic RT-PCR protocol. Each individual chapter in this volume contains complete experimental detail for the protocols described, so that even a newcomer to RT-PCR should be able to perform the techniques. In particular, this volume demonstrates how RT-PCR complements other technologies, such as laser-capture microdissection (LCM), real-time PCR, microarray analysis, high-pressure liquid chromatography (HPLC)and time-resolved fluorometry.
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References
Kumazaki T., Mitsui Y., Hamada K., Sumida H., and Nishiyama M. (1999) Detection of alternative splicing of fibronectin mRNA in a single cell. J. Cell. Sci. 112, 1449–1453.
Sirivatanauksorn Y., Drury R., Crnogorac-Jurcevic T., Sirivatanauksorn V., and Lemoine N.R. (1999) Laser-assisted microdissection:applications in molecular pathology. J. Pathol. 189, 150–154.
Greber-Platzer S., Schatzmann-Turhani D., Cairns N., Balcz B., and Lubec G. (1999) Expression of the transcription factor ETS2 in brain of patients with Down syndrome —evidence against the overexpression-gene dosage hypothesis. J. Neural. Transm. Suppl. 57, 269–281.
Greber-Platzer S., Balcz B., Cairns N., and Lubec G. (1999) c-fos expression in brains of patients with Down syndrome. J. Neural. Transm. Suppl, 57, 75–85.
Goode T., O’Connell J., Ho W.Z., O’Sullivan G.C., Collins J.K., Douglas S. D., et al. (2000) Differential expression of neurokinin-1 receptor by human mucosal and peripheral lymphoid cells. Clin. Diagn. Lab. Immunol. 7, 371–376.
O’Connell J., Goode T., and Shanahan F. (1998) Quantitative measurement of mRNA expression by competitive RT-PCR. Methods Mol. Biol. 92, 183–193.
O’Connell J., Bennett M.W., Nally K., O’Sullivan G.C., Collins J.K., and Shanahan F. (2000) Interferon-gamma sensitizes colonic epithelial cell lines to physiological and therapeutic inducers of colonocyte apoptosis. J. Cell. Physiol. 185, 331–338.
Lai J.P., Douglas S.D., Zhao M., and Ho W.Z. (1999) Quantification of substance P mRNA in human mononuclear phagocytes and lymphocytes using a mimic-based RT-PCR. J. Immunol. Methods. 230, 149–157.
Goode T., O’Connell J., Anton P., Wong H., Reeve J., O’Sullivan G.C., et al. (2000) Neurokinin-1 receptor expression in inflammatory bowel disease:molecular quantitation and localisation. Gut 47, 387–396.
Fanning L., Kenny E., Sheehan M., Cannon B., Whelton M., O’Connell J., et al. (1999) Viral load and clinicopathological features of chronic hepatitis C (1b)in a homogeneous patient population. Hepatology 29, 904–907.
Mullan B., Kenny-Walsh E., Collins J.K., Shanahan F., and Fanning L.J. (2001) Inferred hepatitis C virus quasispecies diversity is influenced by choice of DNA polymerase in reverse transcriptase-polymerase chain reactions. Anal. Biochem. 289, 137–146.
Yeung M.C., Liu J., and Lau A.S. (1996) An essential role for the interferon-inducible, double-stranded RNA-activated protein kinase PKR in the tumor necrosis factor-induced apoptosis in U937 cells. Proc. Natl. Acad. Sci. USA. 93, 12451–12455.
Bachmair F., Huber C., and Daxenbichler G. (1999) Quanititation of gene expression by means of HPLC analysis of RT-PCR products. Clin. Chim. Acta. 279, 25–34.
Halminen M., Sjoroos M., Makela M.J., Waris M., Terho E., Lovgren T., et al. (1999) Simultaneous detection of IFN-gamma and IL-4 mRNAs using RT-PCR and time-resolved fluorometry. Cytokine 11, 87–93.
Sjoroos M., Ilonen J., and Lovgren T. (2001) Solid-phase PCR with hybridization and time-resolved fluorometry for detection of HLA-B27. Clin. Chem. 47, 498–504.
Marchand J., Bord A., Penarier G., Laure F., Carayon P., and Casellas P. (1999) Quantitative method to determine mRNA levels by reverse transcriptase-polymerase chain reaction from leukocyte subsets purified by fluorescence-activated cell sorting:application to peripheral cannabinoid receptors. Cytometry 35, 227–234.
Mann B., Gratchev A., Bohm C., Hanski M.L., Foss H.D., Demel G., et al. (1999) FasL is more frequently expressed in liver metastases of colorectal cancer than in matched primary carcinomas. Br. J. Cancer 79, 1262–1269.
Miyamoto A., Fujiwara Y., Sakon M., Nagano H., Sugita Y., Kondo M., et al. (2000) Development of a multiple-marker RT-PCR assay for detection of micrometastases of hepatocellular carcinoma. Dig. Dis. Sci. 45, 1376–1382.
van der Luijt R., Khan P.M., Vasen H., van Leeuwen C., Tops C., Roest P., et al. (1994) Rapid detection of translation-terminating mutations at the adenomatous polyposis coli (APC)gene by direct protein truncation test. Genomics 20, 1–4.
Kraus C., Gunther K., Vogler A., Hohenberger W., Pfeiffer R.A., and Ballhausen W.G. (1998) Rapid RT-PCR-based protein truncation test in the screening for 5′located mutations of the APC gene. Mol. Cell Probes 12, 143–147.
Waldron-Lynch F., Adams C., Shanahan F., Molloy M.G., and O’Gara F. (1999) Genetic analysis of the 3′untranslated region of the tumour necrosis factor shows a highly conserved region in rheumatoid arthritis affected and unaffected subjects. J. Med. Genet. 36, 214–216.
Luo L., Salunga R.C., Guo H., Bittner A., Joy K.C., Galindo J.E., et al. (1999) Gene expression profiles of laser-captured adjacent neuronal subtypes. Nature Med. 5, 117–122.
Cao Y., Kopplow K., and Liu G.Y. (2000) In-situ immuno-PCR to detect antigens. Lancet 356, 1002–1003.
Bennett M.W., O’Connell J., O’Sullivan G.C., Roche D., Brady C., Kelly J., et al. (1999) Expression of Fas ligand by human gastric adenocarcinomas:a potential mechanism of immune escape in stomach cancer. Gut 44, 156–162.
Kurokawa M., Furukawa H., Yabe T., Matsui T., Toda M., Hamada C., et al. (1999) Frequency of clonally expanded T cells evaluated by PCR from a single cell. J. Immunol. Methods 224, 203–208.
Baert F.J., D’Haens G.R., Peeters M., Hiele M.I., Schaible T.F., Shealy D., et al. (1999) Tumor necrosis factor alpha antibody (infliximab)therapy profoundly downregulates the inflammation in Crohn’s ileocolitis. Gastroenterology 116, 22–28.
Trauth B.C., Klas C., Peters A.M., Matzku S., Moller P., Falk W., et al. (1989) Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science 245, 301–305.
Caron de Fromentel C., Gruel N., Venot C., Debussche L., Conseiller E., Dureuil C., et al. (1999) Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellular expression of anti-p53 single chain Fv fragments. Oncogene 18, 551–557.
Desai S.A., Wang X., Noronha E.J., Kageshita T., and Ferrone S. (1998) Characterization of human anti-high molecular weight melanoma-associated antigen single-chain Fv fragments isolated from a phage display antibody library. Cancer Res. 58, 2417–2425.
Cochet O., Gruel N., Fridman W.H., and Teillaud J.L. (1999) Ras and p53 intracellular targeting with recombinant single-chain Fv (scFv)fragments:a novelapproach for cancer therapy? Cancer Detect.Prev. 23, 506–510.
Tureci O., Sahin U., and Pfreundschuh M. (1997) Serological analysis of human tumor antigens:molecular definition and implications. Mol. Med. Today 3, 342–349.
Healy V., Doonan S., and McCarthy T.V. (1999) Purification, characterization and cDNA cloning of an endo-exonuclease from the basidiomycete fungus Armillaria mellea. Biochem. J. 339, 713–720.
Lambert K.N., and Williamson V.M. (1993) cDNA library construction from small amounts of RNA using paramagnetic beads and PCR. Nucleic Acids Res. 21, 775–776.
Belyavsky A., Vinogradova T., and Rajewsky K. (1989) PCR-based cDNA library construction:general cDNA libraries at the level of a few cells. Nucleic Acids Res. 17, 2919–2932.
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O’Connell, J. (2002). RT-PCR in Biomedicine. In: O’Connell, J. (eds) RT-PCR Protocols. Methods in Molecular Biology, vol 193. Humana Press. https://doi.org/10.1385/1-59259-283-X:003
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DOI: https://doi.org/10.1385/1-59259-283-X:003
Publisher Name: Humana Press
Print ISBN: 978-0-89603-875-2
Online ISBN: 978-1-59259-283-8
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