Quantitative PCR

  • David Sugden
Part of the Springer Protocols Handbooks book series (SPH)


Commonly used methods to quantify RNA and DNA include Northern and Southern blotting, RNase protection assays, and in situ hybridization (see Chapter 29). Because these methods analyze nonamplified RNA or DNA, they are of low sensitivity and require relatively large amounts of nucleic acid. Another method, thousands of times more sensitive than these traditional techniques, combines reverse transcription (RT) and the polymerase chain reaction (PCR). Although RT-PCR is an exquisitely sensitive and specific technique, obtaining quantitative data presents a difficult challenge (1, 2, 3, 4).


Polymerase Chain Reaction Quantitative Polymerase Chain Reaction Molecular Beacon SYBR Green Hydrolysis Probe 
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  1. 1.
    Wang, A. M., Doyle, M. V., and Mark, D. F. (1989) Quantitation of mRNA by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 86, 9717–9721.PubMedCrossRefGoogle Scholar
  2. 2.
    Foley, K. P., Leonard, M. W., and Engel, J. D. (1993) Quantitation of RNA using the polymerase chain reaction. Trends Genet. 9, 380–385.PubMedCrossRefGoogle Scholar
  3. 3.
    Eidne, K.A. (1991) The polymerase reaction and its uses in endocrinology. Trends Endocr. Med. 2, 69–175.Google Scholar
  4. 4.
    Becker-Andre, M. and Hahlbrock, K. (1989) Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY). Nucleic Acids Res. 17, 9437–9446.PubMedCrossRefGoogle Scholar
  5. 5.
    McDowell, D. G., Burns, N. A., and Parkes, H. C. (1998) Localised sequence regions possessing high melting temperatures prevent the amplification of a DNA mimic in competitive PCR. Nucleic Acids Res. 26, 3340–3347.PubMedCrossRefGoogle Scholar
  6. 6.
    Wiesner, R. J. (1992) Direct quantification of picomolar concentrations of mRNAs by mathematical analysis of a reverse transcription/exponential polymerase chain reaction assay. Nucleic Acids Res. 20, 5863–5864.PubMedCrossRefGoogle Scholar
  7. 7.
    Freeman, W. M., Walker, S. J., and Vrana, K. E. (1999) Quantitative RT-PCR: pitfalls and potential. Biotechniques 26, 112–122, 124-125.PubMedGoogle Scholar
  8. 8.
    Kainz, P. (2000) The PCR plateau phase-towards an understanding of its limitations. Biochim. Biophys. Acta 1494, 23–27.PubMedGoogle Scholar
  9. 9.
    Ririe, K. M., Rasmussen, R. P., and Wittwer, C. T. (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 245, 154–160.PubMedCrossRefGoogle Scholar
  10. 10.
    Schneeberger, C., Speiser, P., Kury, F., and Zeillinger, R. (1995) Quantitative detection of reverse transcriptase-PCR products by means of a novel and sensitive DNA stain. PCR Methods Appl. 4, 234–238.PubMedGoogle Scholar
  11. 11.
    Noonan, K. E., Beck, C., Holzmayer, T. A., et al. (1990) Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction. Proc. Natl. Acad. Sci. USA 87, 7160–7164.PubMedCrossRefGoogle Scholar
  12. 12.
    Murphy, L. D., Herzog, C. E., Rudick, J. B., Fojo, A.T., and Bates, S. E. (1990) Use of the polymerase chain reaction in the quantitation of mdr-1 gene expression. Biochemistry 29, 10,351–10,356.PubMedCrossRefGoogle Scholar
  13. 13.
    Kinoshita, T., Imamura, J., Nagai, H., and Shimotohno, K. (1992) Quantification of gene expression over a wide range by the polymerase chain reaction. Anal. Biochem. 206, 231–235.PubMedCrossRefGoogle Scholar
  14. 14.
    Khan, I., Tabb, T., Garfield, R. E., and Grover, A. K. (1992) Polymerase chain reaction assay of mRNA using 28S rRNA as internal standard. Neurosci. Lett. 147, 114–117.PubMedCrossRefGoogle Scholar
  15. 15.
    Siebert, P. D. and Fukuda, M. (1985) Induction of cytoskeletal vimentin and actin gene expression by a tumor-promoting phorbol ester in the human leukemic cell line K562. J. Biol. Chem. 260, 3868–3874.PubMedGoogle Scholar
  16. 16.
    Shinohara, M. L., Loros, J. J., and Dunlap, J. C. (1998) Glyceraldehyde-3-phosphate dehydrogenase is regulated on a daily basis by the circadian clock. J. Biol. Chem. 273, 446–452.PubMedCrossRefGoogle Scholar
  17. 17.
    Siebert, P. D. and Larrick, J.W. (1992) Competitive PCR. Nature 359, 557–558.PubMedCrossRefGoogle Scholar
  18. 18.
    Uberla, K., Platzer, C., Diamantstein, T., and Blankenstein, T. (1991) Generation of competitor DNA fragments for quantitative PCR. PCR Methods Applic. 1, 136–139.Google Scholar
  19. 19.
    Morrison, T. B., Weis, J. J., and Wittwer, C. T. (1998) Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 24, 954–958, 960, 962.PubMedGoogle Scholar
  20. 20.
    Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H. (1991) Detection of specific polymerase chain reaction product by utilizing the 5′→3′-exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88, 7276–7280.PubMedCrossRefGoogle Scholar
  21. 21.
    Tombline, G., Bellizzi, D., and Sgaramella, V. (1996) Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endonuclease activity of DNA polymerases and to premature stops. Proc. Natl. Acad. Sci. USA 93, 2724–2728.PubMedCrossRefGoogle Scholar
  22. 22.
    Tyagi, S. and Kramer, F. R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nature Biotechnol. 14, 303–308.CrossRefGoogle Scholar
  23. 23.
    Whitcombe, D., Theaker, J., Guy, S. P., Brown, T., and Little, S. (1999) Detection of PCR products using self-probing amplicons and fluorescence. Nature Biotechnol. 17, 804–807.CrossRefGoogle Scholar
  24. 24.
    Lowe, B., Avila, H. A., Bloom, F. R., Gleeson, M., and Kusser, W. (2003) Quantitation of gene expression in neural precursors by reverse-transcription polymerase chain reaction using selfquenched, fluorogenic primers. Anal. Biochem. 315, 95–105.PubMedCrossRefGoogle Scholar
  25. 25.
    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 Res. 28, 3752–3761.PubMedCrossRefGoogle Scholar
  26. 26.
    Nazarenko, I., Lowe, B., Darfler, M., Ikonomi, P., Schuster, D., and Rashtchian, A. (2002) Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore. Nucleic Acids Res. 30, e37.PubMedCrossRefGoogle Scholar
  27. 27.
    Nazarenko, I., Pires, R., Lowe, B., Obaidy, M., and Rashtchian, A. (2002) Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes. Nucleic Acids Res. 30, 2089–2195.PubMedCrossRefGoogle Scholar
  28. 28.
    Peirson, S. N., Butler, J. N., and Foster, R. G. (2003) Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Nucleic Acids Res. 31, e73.PubMedCrossRefGoogle Scholar
  29. 29.
    Wilhelm, J., Pingoud, A., and Hahn, M. (2003) Validation of an algorithm for automatic quantification of nucleic acid copy numbers by real-time polymerase chain reaction. Anal. Biochem. 317, 218–225.PubMedCrossRefGoogle Scholar
  30. 30.
    Bustin, S. A. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, 169–193.PubMedCrossRefGoogle Scholar
  31. 31.
    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
  32. 32.
    Vandesompele, J., De Preter, K., Pattyn, F., et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, RESEARCH0034.Google Scholar
  33. 33.
    Goidin, D., Mamessier, A., Staquet, M. J., Schmitt, D., and Berthier-Vergnes, O. (2001) Ribosomal 18S RNA prevails over glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations. Anal. Biochem. 295, 17–21.PubMedCrossRefGoogle Scholar
  34. 34.
    Schmittgen T. D. and Zakrajsek, B.A. (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J. Biochem. Biophys. Methods 46, 69–81.PubMedCrossRefGoogle Scholar
  35. 35.
    Solanas, M., Moral, R., and Escrich, E. (2001) Unsuitability of using ribosomal RNA as loading control for Northern blot analyses related to the imbalance between messenger and ribosomal RNA content in rat mammary tumors. Anal. Biochem. 288, 99–102.PubMedCrossRefGoogle Scholar
  36. 36.
    Jones, L. J., Yue, S. T., Cheung, C. Y., and Singer, V. L. (1998) RNA quantitation by fluorescencebased solution assay: RiboGreen reagent characterization. Anal. Biochem. 265, 368–374.PubMedCrossRefGoogle Scholar
  37. 37.
    Gundry, C. N., Bernard, P. S., Herrmann, M. G., Reed, G. H., and Wittwer, C. T. (1999) Rapid F508del and F508C assay using fluorescent hybridization probes. Genet. Test. 3, 365–370.PubMedGoogle Scholar
  38. 38.
    von Ahsen, N., Oellerich, M., and Schutz, E. (2000) Use of two reporter dyes without interference in a single-tube rapid-cycle PCR: alpha(1)-antitrypsin genotyping by multiplex real-time fluorescence PCR with the LightCycler. Clin. Chem. 46, 156–161.Google Scholar
  39. 39.
    Nauck, M., Marz, W., and Wieland, H. (2000) Evaluation of the Roche diagnostics LightCycler-Factor V Leiden Mutation Detection Kit and the LightCycler-Prothrombin Mutation Detection Kit. Clin. Biochem. 33, 213–216.PubMedCrossRefGoogle Scholar
  40. 40.
    Nauck, M., Hoffmann, M. M., Wieland, H., and Marz, W. (2000) Evaluation of the apo E genotyping kit on the LightCycler. Clin. Chem. 46, 722–724.PubMedGoogle Scholar
  41. 41.
    Mangasser-Stephan, K., Tag, C., Reiser, A. and Gressner, A.M. (1999) Rapid genotyping of hemochromatosis gene mutations on the LightCycler with fluorescent hybridization probes. Clin. Chem. 45, 1875–1878.PubMedGoogle Scholar
  42. 42.
    Fujii, K., Matsubara, Y., Akanuma, J., et al. (2000) Mutation detection by TaqMan-allele specific amplification: application to molecular diagnosis of glycogen storage disease type Ia and mediumchain acyl-CoA dehydrogenase deficiency. Hum. Mutat. 15, 189–196.PubMedCrossRefGoogle Scholar
  43. 43.
    Hiratsuka, M., Agatsuma, Y., Omori, F., et al. (2000) High throughput detection of drug-metabolizing enzyme polymorphisms by allele-specific fluorogenic 5' nuclease chain reaction assay. Biol. Pharm. Bull. 23, 1131–1135.PubMedGoogle Scholar
  44. 44.
    Bon, M. A., van Oeveren-Dybicz, A., and van den Bergh, F. A. (2000) Genotyping of HLA-B27 by real-time PCR without hybridization probes. Clin. Chem. 46, 1000–1002.PubMedGoogle Scholar
  45. 45.
    Wittwer, C. T., Reed, G. H., Gundry, C. N., Vandersteen, J. G., and Pryor, R. J. (2003) High-resolution genotyping by amplicon melting analysis using LCGreen. Clin. Chem. 49, 853–860.PubMedCrossRefGoogle Scholar
  46. 46.
    Gundry, C. N., Vandersteen, J. G., Reed, G. H., Pryor, R. J., Chen, J., and Wittwer, C.T. (2003) Amplicon melting analysis with labeled primers: a closed-tube method for differentiating homozygotes and heterozygotes. Clin. Chem. 49, 396–406.PubMedCrossRefGoogle Scholar
  47. 47.
    Schutten, M. and Niesters H. G. (2001) Clinical utility of viral quantification as a tool for disease monitoring. Expert Rev. Mol. Diagn. 1, 53–62.CrossRefGoogle Scholar
  48. 48.
    Niesters, H. G. (2002) Clinical virology in real time. J. Clin. Virol. 25(Suppl 3), S3–12.PubMedCrossRefGoogle Scholar
  49. 49.
    Mackay, I. M., Arden, K. E., and Nitsche, A. (2002) Real-time PCR in virology. Nucleic Acids Res. 30, 1292–1305.PubMedCrossRefGoogle Scholar
  50. 50.
    Carpenter, C. C., Cooper, D. A., Fischl, M. A., et al. (2000) Antiretroviral therapy in adults: updated recommendations of the International AIDS Society-USA Panel. JAMA 283, 381–390.PubMedCrossRefGoogle Scholar
  51. 51.
    Kelley, V. A. and Caliendo, A. M. (2001) Successful testing protocols in virology. Clin. Chem. 47, 1559–1562.PubMedGoogle Scholar
  52. 52.
    Berger, A. and Preiser, W. (2002) Viral genome quantification as a tool for improving patient management: the example of HIV, HBV, HCV and CMV. J. Antimicrob. Chemother. 49, 713–721.PubMedCrossRefGoogle Scholar
  53. 53.
    Schweiger, B., Zadow, I., Heckler, R., Timm, H., and Pauli, G. (2000) Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples. J. Clin. Microbiol. 38, 1552–1558.PubMedGoogle Scholar
  54. 54.
    Rantakokko-Jalava, K. and Jalava, J. (2001) Development of conventional and real-time PCR assays for detection of Legionella DNA in respiratory specimens. J. Clin. Microbiol. 39, 2904–2910.PubMedCrossRefGoogle Scholar
  55. 55.
    Elsayed, S., Chow, B. L., Hamilton, N. L, Gregson, D. B., Pitout, J. D., and Church, D. L. (2003) Development and validation of a molecular beacon probe-based real-time polymerase chain reaction assay for rapid detection of methicillin resistance in Staphylococcus aureus. Arch. Pathol. Lab. Med. 127, 845–849.Google Scholar
  56. 56.
    Palladino, S., Kay, I. D., Flexman, J. P., et al. (2003) Rapid detection of vanA and vanB genes directly from clinical specimens and enrichment broths by real-time multiplex PCR assay. J. Clin. Microbiol. 41, 2483–2486.PubMedCrossRefGoogle Scholar
  57. 57.
    White, P. L., Shetty, A., and Barnes, R. A. (2003) Detection of seven Candida species using the Light-Cycler system. J. Med. Microbiol. 52, 229–238.PubMedCrossRefGoogle Scholar
  58. 58.
    Chen, S., Yee, A., Griffiths, M., et al. (1997) The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities. Int. J. Food Microbiol. 35, 239–250.PubMedCrossRefGoogle Scholar
  59. 59.
    Chen, W., Martinez, G., and Mulchandani, A. (2000) Molecular beacons: a real-time polymerase chain reaction assay for detecting Salmonella. Anal. Biochem. 280, 166–172.CrossRefGoogle Scholar
  60. 60.
    Bhagwat, A. A. (2003) Simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains by real-time PCR. Int. J. Food Microbiol. 84, 217–224.PubMedGoogle Scholar
  61. 61.
    Jaeger, U. and Kainz, B. (2003) Monitoring minimal residual disease in AML: the right time for real time. Ann. Hematol. 82, 139–147.PubMedGoogle Scholar
  62. 62.
    Estalilla, O. C., Medeiros, L. J., Manning, J. T. Jr., and Luthra, R. (2000) 5′→3′ exonuclease-based real-time PCR assays for detecting the t(14;18)(q32;21): a survey of 162 malignant lymphomas and reactive specimens. Mod. Pathol. 13, 661–666.PubMedCrossRefGoogle Scholar
  63. 63.
    Barthe, C., Mahon, F. X., Gharbi, M. J., et al. (2001) Expression of interferon-alpha (IFN-alpha) receptor 2c at diagnosis is associated with cytogenetic response in IFN-alpha-treated chronic myeloid leukemia. Blood 97, 3568–3573.PubMedCrossRefGoogle Scholar
  64. 64.
    Ginzinger, D. G. (2002) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp. Hematol. 30, 503–512.PubMedCrossRefGoogle Scholar
  65. 65.
    van der Velden, V. H., Hochhaus, A., Cazzaniga, G., Szczepanski, T., Gabert, J., and van Dongen, J. J. (2003) Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects. Leukemia 17, 1013–1034.PubMedCrossRefGoogle Scholar
  66. 66.
    Mocellin, S., Rossi, C. R., Pilati, P., Nitti, D., and Marincola, F.M. (2003) Quantitative real-time PCR: a powerful ally in cancer research. Trends Mol. Med. 9, 189–195.PubMedCrossRefGoogle Scholar
  67. 67.
    Bockmann, B., Grill, H. J., and Giesing, M. (2001) Molecular characterization of minimal residual cancer cells in patients with solid tumors. Biomol. Eng. 17, 95–111.PubMedCrossRefGoogle Scholar
  68. 68.
    Ahmed, F. E. (2002) Detection of genetically modified organisms in foods. Trends Biotechnol. 20, 215–223.PubMedCrossRefGoogle Scholar
  69. 69.
    Beachy, R. N. (1999) Facing fear of biotechnology. Science 285, 335.PubMedCrossRefGoogle Scholar
  70. 70.
    Vaitilingom, M., Pijnenburg, H., Gendre, F., and Brignon, P. (1999) Real-time quantitative PCR detection of genetically modified Maximizer maize and Roundup Ready soybean in some representative foods. J. Agric. Food Chem. 47, 5261–5266.PubMedCrossRefGoogle Scholar
  71. 71.
    Permingeat, H. R., Reggiardo, M. I., and Vallejos, R. H. (2002) Detection and quantification of transgenes in grains by multiplex and real-time PCR. J. Agric. Food Chem. 50, 4431–4436.PubMedCrossRefGoogle Scholar
  72. 72.
    Broussard, L. A. (2001) Biological agents: weapons of warfare and bioterrorism. Mol. Diagn. 6, 323–333.PubMedGoogle Scholar
  73. 73.
    Lee, M. A., Brightwell, G., Leslie, D., Bird, H., and Hamilton, A. (1999) Fluorescent detection techniques for real-time multiplex strand specific detection of Bacillus anthracis using rapid PCR. J. Appl. Microbiol. 87, 218–223.PubMedCrossRefGoogle Scholar
  74. 74.
    Bell, C. A., Uhl, J. R., Hadfield, T. L., et al. (2002) Detection of Bacillus anthracis DNA by LightCycler PCR. J. Clin. Microbiol. 40, 2897–2902.PubMedCrossRefGoogle Scholar
  75. 75.
    Ibrahim, M. S., Kulesh, D. A., Saleh, S. S., et al. (2003) Real-time PCR assay to detect smallpox virus. J. Clin. Microbiol. 41, 3835–3839.CrossRefGoogle Scholar
  76. 76.
    Higgins, J. A., Ezzell, J., Hinnebusch, B. J., Shipley, M., Henchal, E. A., and Ibrahim, M. S. (1998) 5′ nuclease PCR assay to detect Yersinia pestis. J. Clin. Microbiol. 36, 2284–2288.PubMedGoogle Scholar
  77. 77.
    Higgins, J. A., Hubalek, Z., Halouzka, J., et al. (2000) Detection of Francisella tularensis in infected mammals and vectors using a probe-based polymerase chain reaction. Am. J. Trop. Med. Hyg. 62, 310–318.PubMedGoogle Scholar
  78. 78.
    Drosten, C., Gottig, S., Schilling, S., et al. (2002) Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR. J. Clin. Microbiol. 40, 2323–2330PubMedCrossRefGoogle Scholar
  79. 79.
    Higgins, J. A., Nasarabadi, S., Karns, J. S., et al. (2003) A handheld real time thermal cycler for bacterial pathogen detection. Biosens. Bioelectron. 18, 1115–1123.PubMedCrossRefGoogle Scholar
  80. 80.
    McCartney, H. A., Foster, S. J., Fraaije, B. A., and Ward, E. (2003) Molecular diagnostics for fungal plant pathogens. Pest Manag. Sci. 59, 129–142.PubMedCrossRefGoogle Scholar
  81. 81.
    Wetton, J. H., Tsang, C. S., Roney, C. A., and Spriggs, A. C. (2002) An extremely sensitive speciesspecific ARMS PCR test for the presence of tiger bone DNA. Forensic Sci. Int. 126, 137–144.PubMedCrossRefGoogle Scholar
  82. 82.
    Siva, S. C., Johnson, S. I., McCracken, S. A., and Morris, J. M. (2003) Evaluation of clinical usefulness of isolation of fetal DNA from the maternal circulation. Aust. NZ J. Obstet. Gyneacol. 43, 10–15CrossRefGoogle Scholar
  83. 83.
    Costa, J. M., Giovangrandi, Y., Ernault, P., et al. (2002) Fetal RHD genotyping in maternal serum during the first trimester of pregnancy. Br. J. Haematol. 119, 255–260.PubMedCrossRefGoogle Scholar
  84. 84.
    Costa, J. M., Benachi, A., Olivi, M., Dumez, Y., Vidaud, M., and Gautier, E. (2003) Fetal expressed gene analysis in maternal blood: a new tool for noninvasive study of the fetus. Clin. Chem. 49, 981–983.PubMedCrossRefGoogle Scholar
  85. 85.
    Lo, Y. M. D., Leung, T. N., Tein, M. S. C., et al. (1999) Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin. Chem. 45, 184–188.PubMedGoogle Scholar
  86. 86.
    Ng, E. K., Tsui, N. B., Lau, T. K., et al. (2003) mRNA of placental origin is readily detectable in maternal plasma. Proc. Natl. Acad. Sci. USA 100, 4748–4753.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • David Sugden
    • 1
  1. 1.Centre for Reproduction, Endocrinology, and Diabetes, School of Biomedical SciencesKing’s College LondonLondonUK

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