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Quantitative TaqMan Real-Time PCR

Diagnostic and Scientific Applications
  • Jörg Dötsch
  • Ellen Schoof
  • Wolfgang Rascher
Protocol
  • 1.5k Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

The invention of real-time polymerase chair reaction (PCR) has revolutionized the quantification of gene expression and DNA copy number measurements. However, after the first documentation of real-time PCR in 1993 (1), it took several years for this method to become a mainstream tool. PCR generates DNA copies in an exponential way. As soon as resources are exhausted, however, the so-called plateau phase of PCR reaction is reached, making quantification very unreliable. Therefore, quantification appears most reliable in the early exponential phase of PCR (i.e., in a “real-time” fashion). To ensure measurements in this phase of the PCR cycle, real-time PCR measures as soon as the threshold of detection is definitely reached. The cycle of PCR at which this occurs is then named the threshold cycle (2) (see Fig. 1). It is the objective of this chapter to describe the possibilities of TaqMan real-time PCR for mRNA and DNA quantification and to discuss pitfalls and alternatives.

Keywords

Minimal Residual Disease TaqMan Probe Molecular Beacon MYCN Amplification Cytogenetic Aberration 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Higuchi, R., Fockler, C., Dollinger, G., and Watson R. (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 11, 1026–1030.PubMedCrossRefGoogle Scholar
  2. 2.
    Ginzinger, D. G. (1993) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp. Hematol. 30, 503–512.CrossRefGoogle Scholar
  3. 3.
    Dötsch, J., Repp, R., Rascher, W., and Christiansen, H. (2001) Diagnostic and scientific applications of TaqMan real-time PCR in neuroblastomas. Expert Rev. Mol. Diagn. 1, 233–238.PubMedCrossRefGoogle Scholar
  4. 4.
    Dötsch, J., Nüsken, K. D., Knerr, I., Kirschbaum, M., Repp, R., and Rascher, W. (1999) Leptin and neuropeptide Y gene expression in human placenta: ontogeny and evidence for similarities to hypothalamic regulation. J. Clin. Endocrinol. Metab. 84, 2755–2758.PubMedCrossRefGoogle Scholar
  5. 5.
    Orlando, C., Pinzani, P., and Pazzaggli, M. (1998) Developments in quantitative PCR. Clin. Chem. Lab. Med. 36, 255–269.PubMedCrossRefGoogle Scholar
  6. 6.
    Dötsch, J., Harmjanz, A., Christiansen, H., Hänze, J., Lampert, F., and Rascher, W. (2000) Gene expression of neuronal nitric oxide synthase and adrenomedullin in human neuroblastoma using real-time PCR. Int. J. Cancer 88, 172–175.PubMedCrossRefGoogle Scholar
  7. 7.
    Förster, E. (1994) Rapid generation of internal standards for competitive PCR by low-stringency primer annealing. BioTechniques 16, 1006–1008.PubMedGoogle Scholar
  8. 8.
    Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M. (1996) Real-time quantitative PCR. Genome Res. 6, 986–994.PubMedCrossRefGoogle Scholar
  9. 9.
    Gibson, U. E., Heid, C. A., and Williams, P. M. (1996) A novel method for real-time quantitative RT-PCR. Genome Res. 6, 995–1001.PubMedCrossRefGoogle Scholar
  10. 10.
    Raggi, C. C., Bagnoni, M. L., Tonini, G. P., et al. (1999) Real-time quantitative PCR for the measurement of MYCN amplification in human neuroblastoma with the TaqMan detection system. Clin. Chem. 45, 1918–1924.PubMedGoogle Scholar
  11. 11.
    Klein, D. (2002) Quantification using real-time PCR technology: applications and limitations. Trends Mol. Med. 8, 257–260.PubMedCrossRefGoogle Scholar
  12. 12.
    Bustin, S. A. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, 16–193.CrossRefGoogle Scholar
  13. 13.
    Schoof, E., Girstl, M., Frobenius, W., et al. (2001) Decreased gene expression of 11betahydroxysteroid dehydrogenase type 2 and 15-hydroxyprostaglandin dehydrogenase in human placenta of patients with preeclampsia. J. Clin. Endocrinol. Metab. 86, 1313–1317.PubMedCrossRefGoogle Scholar
  14. 14.
    Schoof, E., Stuppy, A., Harig, F., et al. (2003) No influence of surgical stress on postoperative leptin gene expression in different adipose tissues and soluble leptin receptor plasma levels. Horm. Res. 59, 184–190.PubMedCrossRefGoogle Scholar
  15. 15.
    Trollmann, R., Amann, K., Schoof, E., et al. (2003) Hypoxia activates the human placental vascular endothelial growth factor system in vitro and in vivo: up-regulation of vascular endothelial growth factor in clinically relevant hypoxic ischemia in birth asphyxia. Am. J. Obstet. Gynecol. 188, 517–523.PubMedCrossRefGoogle Scholar
  16. 16.
    Brodeur, G. M., Seeger, R. C., Schwab, M., Varmus, H. E., and Bishop, J. M. (1984) Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science 224, 1121–1124.PubMedCrossRefGoogle Scholar
  17. 17.
    Seeger, R. C., Brodeur, G. M., Sather, H., et al. (1985) Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N. Engl. J. Med. 313, 1111–1116.PubMedCrossRefGoogle Scholar
  18. 18.
    Pession, A., Trere, D., Perri, P., et al. (1997) N-myc amplification and cell proliferation rate in human neuroblastoma. J. Pathol. 183, 339–344.PubMedCrossRefGoogle Scholar
  19. 19.
    De Cremoux, P., Thioux, M., Peter, M., et al. (1997) Polymerase chain reaction compared with dot blotting for the determination of N-myc gene amplification in neuroblastoma. Int. J. Cancer 72, 518–521.PubMedCrossRefGoogle Scholar
  20. 20.
    Sestini, R., Orlando, C., Zentilin, L., et al. (1995) Gene amplification for c-erbB-2, c-myc, epidermal growth factor receptor, int-2, and N-myc measured by quantitative PCR with a multiple competitor template. Clin. Chem. 41, 826–832.PubMedGoogle Scholar
  21. 21.
    Shapiro, D. N., Valentine, M. B., Rowe, S. T., et al. (1993) Detection of N-myc gene amplification by fluorescence in situ hybridization. Diagnostic utility for neuroblastoma. Am. J. Pathol. 142, 1339–1346.PubMedGoogle Scholar
  22. 22.
    Kwan, E., Norris, M. D., Zhu, L., Ferrara, D., Marshall, G. M., and Haber, M. (2000) Simultaneous detection and quantification of minimal residual disease in childhood acute lymphoblastic leukaemia using real-time polymerase chain reaction. Br. J. Haematol. 109, 430–434.PubMedCrossRefGoogle Scholar
  23. 23.
    Cilloni, D., Gottardi, E., De Micheli, D., et al. (2002) Quantitative assessment of WT1 expression by real-time quantitative PCR may be a useful tool for monitoring minimal residual disease in acute leukemia patients. Leukemia 16, 2115–2121.PubMedCrossRefGoogle Scholar
  24. 24.
    Brinkschmidt, C., Christiansen, H., Terpe, H. J., et al. (1997) Comparative genomic hybridization (CGH) analysis of neuroblastomas-an important methodological approach in pediatric tumour pathology. J. Pathol. 181, 394–400.PubMedCrossRefGoogle Scholar
  25. 25.
    Brunk, C. F., Li, J., Avaniss-Aghajani, E. (2002) Analysis of specific bacteria from environmental samples using a quantitative polymerase chain reaction. Curr Issues Mol Biol. 4, 13–18.Google Scholar
  26. 26.
    Klein, D., Leutenegger, C. M., Bahula, C., et al. (2001) Influence of preassay and sequence variations on viral load determination by a multiplex real-time reverse transcriptase-polymerase chain reaction for feline immunodeficiency virus. J. Acquired Immune Defic. Syndr. 26, 8–20.Google Scholar
  27. 27.
    Gruber, F., Falkner, F. G., Dorner, F., and Hammerle, T. (2001) Quantitation of viral DNA by real-time PCR applying duplex amplification, internal standardization, and two-color fluorescence detection. Appl. Environ. Microbiol. 67, 2837–2839.PubMedCrossRefGoogle Scholar
  28. 28.
    Bustin S. A. (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol. 29, 23–39.PubMedCrossRefGoogle Scholar
  29. 29.
    Wittwer, C., Herrmann, M., Moss, A., and Rasmussen, R. (1997) Continuous fluorescence monitoring of rapid cycle DNA amplification. BioTechniques 22, 130–138.PubMedGoogle Scholar
  30. 30.
    Giulietti, A., Overbergh, L., Valckx, D., Decallonne, B., Bouillon, R., and Mathieu, C. (2001) An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25, 386–401.PubMedCrossRefGoogle Scholar
  31. 31.
    Tyagi, S. and Kramer, R. (1996) Molecular beacons: probes that fluorescence upon hybridization. Nature Biotechnol. 14, 303–308.CrossRefGoogle Scholar
  32. 32.
    Zubritsky, E. (1999) Widespread interest in gene quantification and high-throughput assays are putting quantitative PCR back in the spotlight. Anal. Chem. 71, 191A–195A.Google Scholar
  33. 33.
    Nakagawara, A., Arima-Nakagawara, M., Scavarda, N. J., Azar, C. G., Cantor, A. B., and Brodeur, G. M. (1993) Association between high levels of expression of the TRK gene and favorable outcome in human neuroblastoma. N. Engl. J. Med. 328, 847–854.PubMedCrossRefGoogle Scholar
  34. 34.
    Chan, H. S., Haddad, G., Thorner, P. S., et al. (1991) P-Glycoprotein expression as a predictor of the outcome of therapy for neuroblastoma. N. Engl. J. Med. 325, 1608–1614.PubMedCrossRefGoogle Scholar
  35. 35.
    Norris, M. D., Bordow, S. B., Marshall, G. M., Haber, P. S., Cohn, S. L., and Haber, M. (1996) Expression of the gene for multidrug-resistance-associated protein and outcome in patients with neuroblastoma. N. Engl. J. Med. 334, 231–238.PubMedCrossRefGoogle Scholar
  36. 36.
    Leone, A., Seeger, R. C., Hong, C. M., et al. (1993) Evidence for nm23 RNA overexpression, DNA amplification and mutation in aggressive childhood neuroblastomas. Oncogene 8, 855–865.PubMedGoogle Scholar
  37. 37.
    Favrot, M. C., Combaret, V., and Lasset, C. (1993) CD44-a new prognostic marker for neuroblastoma. N. Engl. J. Med. 329, 1965.PubMedCrossRefGoogle Scholar
  38. 38.
    Hahn, S., Zhong, X. Y., Burk, M. R., Troeger, C., and Holzgreve, W. (2000) Multiplex and real-time quantitative PCR on fetal DNA in maternal plasma. A comparison with fetal cells isolated from maternal blood. Ann. NY Acad. Sci. 906, 148–152.PubMedCrossRefGoogle Scholar
  39. 39.
    Von Ahsen, N., Oellerich, M., and Schutz, E. (2000) A method for homogeneous color-compensated genotyping of factor V (G1691A) and methylenetetrahydrofolate reductase (C677T) mutations using real-time multiplex fluorescence PCR. Clin. Biochem. 33, 535–539.CrossRefGoogle Scholar
  40. 40.
    Norris, M. D., Burkhart, C. A., Marshall, G. M., Weiss, W. A., and Haber, M. (2000) Expression of N-myc and MRP genes and their relationship to N-myc gene dosage and tumor formation in a murine neuroblastoma model. Med. Pediatr. Oncol. 35, 585–589.PubMedCrossRefGoogle Scholar
  41. 41.
    Kawamoto, T., Shishikura, T., Ohira, M., et al. (2000) Association between favorable neuroblastoma and high expression of the novel metalloproteinase gene, nbla3145/XCE, cloned by differential screening of the full-length-enriched oligo-capping neuroblastoma cDNA libraries. Med. Pediatr. Oncol. 35, 628–631.PubMedCrossRefGoogle Scholar
  42. 42.
    Fink, L., Seeger, W., Ermert, L., et al. (1998) Real-time quantitative RT-PCR after laser-assisted cell picking. Nature Med. 4, 1329–1333.PubMedCrossRefGoogle Scholar
  43. 43.
    von der Hardt, K., Kandler, M. A., Fink, L., et al. Laser-assisted microdissection and real-time PCR detect anti-inflammatory effect of perfluorocarbon. Am. J. Physiol. Lung Cell Mol. Physiol. 285, L55–L62.Google Scholar
  44. 44.
    Lehmann, U. and Kreipe, H. (2001) Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies. Methods 2, 409–418.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • Jörg Dötsch
    • 1
  • Ellen Schoof
    • 1
  • Wolfgang Rascher
    • 1
  1. 1.Department of PediatricsUniversity of ErlangenErlangenGermany

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