Abstract
Analysis of RNA expression levels by real-time reverse-transcription (RT) PCR has become a routine technique in diagnostic and research laboratories. Monitoring of DNA amplification can be done using fluorescent sequence-specific probes, which generate signal only upon binding to their target. Numerous fluorescent dyes with unique emission spectra are available and can be used to differentially label probes for various genes. Such probes can be added to the same PCR amplification reaction for simultaneous detection of multiple targets in a single assay. Such multiplexing is advantageous, since it markedly increases throughput and decreases costs and labor. Here, we describe application of multiplex real-time RT-PCR using TaqMan probes in the analysis of relative expression levels of a novel tumor-associated gene CUG2 in cell lines and tissue samples.
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References
Murphy, J. and Bustin, S.A. (2009) Reliability of real-time reverse-transcription PCR in clinical diagnostics: gold standard or substandard? Expert Review of Molecular Diagnostics, 9, 187–197.
Schmittgen, T.D., Lee, E.J., Jiang, J., Sarkar, A., Yang, L., Elton, T.S. and Chen, C. (2008) Real-time PCR quantification of precursor and mature microRNA. Methods (San Diego, Calif), 44, 31–38.
Koga, T., Tokunaga, E., Sumiyoshi, Y., Oki, E., Oda, S., Takahashi, I., Kakeji, Y., Baba, H. and Maehara, Y. (2008) Detection of circulating gastric cancer cells in peripheral blood using real time quantitative RT-PCR. Hepato-gastroenterology, 55, 1131–1135.
Rizzi, F., Belloni, L., Crafa, P., Lazzaretti, M., Remondini, D., Ferretti, S., Cortellini, P., Corti, A. and Bettuzzi, S. (2008) A novel gene signature for molecular diagnosis of human prostate cancer by RT-qPCR. PloS One, 3, e3617.
Pignot, G., Bieche, I., Vacher, S., Guet, C., Vieillefond, A., Debre, B., Lidereau, R. and Amsellem-Ouazana, D. (2009) Large-scale real-time reverse transcription-PCR approach of angiogenic pathways in human transitional cell carcinoma of the bladder: identification of VEGFA as a major independent prognostic marker. European Urology, 56, 678–688.
Andreeff, M., Ruvolo, V., Gadgil, S., Zeng, C., Coombes, K., Chen, W., Kornblau, S., Baron, A.E. and Drabkin, H.A. (2008) HOX expression patterns identify a common signature for favorable AML. Leukemia, 22, 2041–2047.
Wakeley, P.R., Johnson, N., McElhinney, L.M., Marston, D., Sawyer, J. and Fooks, A.R. (2005) Development of a real-time, TaqMan reverse transcription-PCR assay for detection and differentiation of lyssavirus genotypes 1, 5, and 6. Journal of Clinical Microbiology, 43, 2786–2792.
Lu, X., Holloway, B., Dare, R.K., Kuypers, J., Yagi, S., Williams, J.V., Hall, C.B. and Erdman, D.D. (2008) Real-time reverse transcription-PCR assay for comprehensive detection of human rhinoviruses. Journal of Clinical Microbiology, 46, 533–539.
Wang, L., Giannoudis, A., Lane, S., Williamson, P., Pirmohamed, M. and Clark, R.E. (2008) Expression of the uptake drug transporter hOCT1 is an important clinical determinant of the response to imatinib in chronic myeloid leukemia. Clinical Pharmacology and Therapeutics, 83, 258–264.
Mischitelli, M., Fioriti, D., Anzivino, E., Bellizzi, A., Ferretti, G., Gussman, N., Mitterhofer, A.P., Tinti, F., Barile, M., Dal Maso, M. et al.(2007) BKV QPCR detection and infection monitoring in renal transplant recipients. New Microbiol, 30, 271–274.
Mackay, J. and Landt, O. (2007) Real-time PCR fluorescent chemistries. Methods in Molecular Biology (Clifton, N.J) 353, 237–261.
Gibson, U.E., Heid, C.A. and Williams, P.M. (1996) A novel method for real time quantitative RT-PCR. Genome Research, 6, 995–1001.
Tyagi, S. and Kramer, F.R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nature Biotechnology, 14, 303–308.
Whitcombe, D., Theaker, J., Guy, S.P., Brown, T. and Little, S. (1999) Detection of PCR products using self-probing amplicons and fluorescence. Nature Biotechnology, 17, 804–807.
Thelwell, N., Millington, S., Solinas, A., Booth, J. and Brown, T. (2000) Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Research, 28, 3752–3761.
Lee, S., Gang, J., Jeon, S.B., Choo, S.H., Lee, B., Kim, Y.G., Lee, Y.S., Jung, J., Song, S.Y. and Koh, S.S. (2007) Molecular cloning and functional analysis of a novel oncogene, cancer-upregulated gene 2 (CUG2). Biochemical and Biophysical Research Communications, 360, 633–639.
Kim, H., Lee, M., Lee, S., Park, B., Koh, W., Lee, D.J., Lim, D.S. and Lee, S. (2009) Cancer-upregulated gene 2 (CUG2), a new component of centromere complex, is required for kinetochore function. Molecules and Cells, 27, 697–701.
de Kok, J.B., Roelofs, R.W., Giesendorf, B.A., Pennings, J.L., Waas, E.T., Feuth, T., Swinkels, D.W. and Span, P.N. (2005) Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Laboratory Investigation; a Journal of Technical Methods and Pathology, 85, 154–159.
Gresner, P., Gromadzinska, J. and Wasowicz, W. (2009) Reference genes for gene expression studies on non-small cell lung cancer. Acta Biochimica Polonica, 56, 307–316.
Pernot, F., Dorandeu, F., Beaup, C. and Peinnequin, A. (2010) Selection of reference genes for real-time quantitative reverse transcription-polymerase chain reaction in hippocampal structure in a murine model of temporal lobe epilepsy with focal seizures. Journal of Neuroscience Research, 8, 1000–1008.
Taveau, M., Stockholm, D., Spencer, M. and Richard, I. (2002) Quantification of splice variants using molecular beacon or scorpion primers. Analytical Biochemistry, 305, 227–235.
Roux, K.H. (2002) Single-step PCR optimization using touchdown and stepdown PCR programming. Methods in Molecular Biology (Clifton, N.J.), 192, 31–36.
Acknowledgments
Authors would like to thank Alexander Skoblov and Michelle Nyhan for their help and advice in preparation of the manuscript.
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Gurvich, O.L., Skoblov, M. (2011). Real-Time PCR and Multiplex Approaches. In: O'Driscoll, L. (eds) Gene Expression Profiling. Methods in Molecular Biology, vol 784. Humana Press. https://doi.org/10.1007/978-1-61779-289-2_1
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DOI: https://doi.org/10.1007/978-1-61779-289-2_1
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