Real-Time PCR and Multiplex Approaches

  • Olga L. GurvichEmail author
  • Mikhail Skoblov
Part of the Methods in Molecular Biology book series (MIMB, volume 784)


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.

Key words

Multiplex Real-time PCR Gene expression TaqMan probes CUG2 C6orf173 



Authors would like to thank Alexander Skoblov and Michelle Nyhan for their help and advice in preparation of the manuscript.


  1. 1.
    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.PubMedCrossRefGoogle Scholar
  2. 2.
    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.Google Scholar
  3. 3.
    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.PubMedGoogle Scholar
  4. 4.
    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.PubMedCrossRefGoogle Scholar
  5. 5.
    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.PubMedCrossRefGoogle Scholar
  6. 6.
    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.PubMedCrossRefGoogle Scholar
  7. 7.
    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.PubMedCrossRefGoogle Scholar
  8. 8.
    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.PubMedCrossRefGoogle Scholar
  9. 9.
    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.PubMedCrossRefGoogle Scholar
  10. 10.
    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.PubMedGoogle Scholar
  11. 11.
    Mackay, J. and Landt, O. (2007) Real-time PCR fluorescent chemistries. Methods in Molecular Biology (Clifton, N.J) 353, 237–261.Google Scholar
  12. 12.
    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.PubMedCrossRefGoogle Scholar
  13. 13.
    Tyagi, S. and Kramer, F.R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nature Biotechnology, 14, 303–308.PubMedCrossRefGoogle Scholar
  14. 14.
    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.PubMedCrossRefGoogle Scholar
  15. 15.
    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.PubMedCrossRefGoogle Scholar
  16. 16.
    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.PubMedCrossRefGoogle Scholar
  17. 17.
    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.PubMedCrossRefGoogle Scholar
  18. 18.
    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.Google Scholar
  19. 19.
    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.PubMedGoogle Scholar
  20. 20.
    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.Google Scholar
  21. 21.
    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.PubMedCrossRefGoogle Scholar
  22. 22.
    Roux, K.H. (2002) Single-step PCR optimization using touchdown and stepdown PCR ­programming. Methods in Molecular Biology (Clifton, N.J.), 192, 31–36.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Moscow State UniversityMoscowRussia
  2. 2.Research Centre for Medical GeneticsRussian Academy of Medical SciencesMoscowRussia

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