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Monitoring the Immune Response Using Real-Time PCR

  • Protocol
DNA and RNA Profiling in Human Blood

Part of the book series: METHODS IN MOLECULAR BIOLOGY™ ((MIMB,volume 496))

Abstract

Induction of an immune response to a particular antigen is the basis of vaccination. This has been done for years to prevent infectious diseases, and has the potential for the treatment of cancer. The immune response is nowadays more precisely modulated rather than simply induced, like in case of immunotherapy of allergic diseases. Likewise, autoimmune diseases are associated with an inappropriate immune response, and many efforts are made for specifically inhibiting this unwanted response. A possible line of attack is the induction of an antigen-specific immune tolerance, which also has a use in the field of transplantation, where allogeneic responses are deleterious for the graft. In all of these fields of fundamental and clinical medicine, the modulation of immune response requires the assistance of laboratory tests, among which real-time PCR appears more and more helpful. This chapter describes a protocol to quantify immune-related mRNAs using reverse transcription-real-time PCR. The transcripts can be quantified in cultured cells or in cultured whole blood, after an incubation period in the presence of the antigen to which the immune response is analyzed. This is the typical approach to evaluate the efficacy of a vaccine. The transcripts can also be quantified directly in the biological sample, giving information about the in vivo immune status of the individual. The techniques to achieve these different methods are described, and are illustrated by the analysis of the response against the toxoid tetanus antigen.

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References

  1. Bouma, G. J., Schanz, U., Oudshoorn, M., et al. (1996) A cell-saving non-radioactive limiting dilution analysis-assay for the combined determination of helper and cytotoxic T lymphocyte precursor frequencies. Bone Marrow Transplant 17, 19–23.

    CAS  PubMed  Google Scholar 

  2. van Besouw, N. M., Zuijderwijk, J. M., de Kuiper, P., et al. (2005) The granzyme B and interferon-gamma enzyme-linked immunospot assay as alternatives for cytotoxic T-lymphocyte precursor frequency after renal transplantation. Transplantation 79, 1062–1066.

    Article  PubMed  Google Scholar 

  3. Hernandez-Fuentes, M. P., Salama, A. (2006) In vitro assays for immune monitoring in transplantation. Meth Mol Biol 333, 269–290.

    CAS  Google Scholar 

  4. Keilholz, U., Weber, J., Finke, J.H., et al. (2002) Immunologic monitoring of cancer vaccine therapy: results of a workshop sponsored by the Society for Biological Therapy. J Immunother 25, 97–138.

    Article  PubMed  Google Scholar 

  5. Cekaite, L., Hovig, E., Sioud, M. (2007) Protein arrays: a versatile toolbox for target identification and monitoring of patient immune responses. Meth Mol Biol 360,335–348.

    CAS  Google Scholar 

  6. Bustin, S. A., Benes, V., Nolan, T., et al. (2005) Quantitative real-time RT-PCR–a perspective. J Mol Endocrinol 34, 597–601.

    Article  CAS  PubMed  Google Scholar 

  7. Panelli, M. C., Wang, E., Monsurro, V. et al. (2002) The role of quantitative PCR for the immune monitoring of cancer patients. Expert Opin Biol Ther 5, 557–564.

    Google Scholar 

  8. Stordeur, P. (2005) Immune monitoring – Applications of real-time PCR, in Encyclopedia of Medical Genomics and Proteomics (Fuchs J. and Podda M., ed). Dekker encyclopedias, NJ, http://www.dekker.com/sdek/abstract~db=enc~content=a713596205/.

  9. Li, B., Hartono, C., Ding, R., et al. (2001) Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin and granzyme B in urine. N Engl J Med 344, 947–954.

    Article  CAS  PubMed  Google Scholar 

  10. Bustin, S. A. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25, 169–193.

    Google Scholar 

  11. Bustin, S. A. (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29, 23–39.

    Google Scholar 

  12. Wong, M. L., Medrano, J. F. (2005) Real-time PCR for mRNA quantitation. Biotechniques 39, 75–85.

    Article  CAS  PubMed  Google Scholar 

  13. Overbergh, L., Valckx, D., Waer, M., et al. (1999) Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR. Cytokine 11, 305–312.

    Article  CAS  PubMed  Google Scholar 

  14. Stordeur, P., Poulin, L. F., Craciun, L., et al. (2002) Cytokine mRNA quantification by real-time PCR. J Immunol Meth 259,55–64. Erratum in (2002) J Immunol Meth 262: 229.

    Article  CAS  Google Scholar 

  15. Stordeur, P., Zhou, L., Byl, B., et al. (2003) Immune monitoring in whole blood using real-time PCR. J Immunol Meth 276, 69–77.

    Article  CAS  Google Scholar 

  16. Zhou, L., Toungouz, M., Donckier, V., et al. (2005) A rapid test to monitor alloreactive responses in whole blood using real-time polymerase chain reaction. Transplantation 80, 410–413.

    Article  CAS  PubMed  Google Scholar 

  17. Craciun, L., Stordeur, P., Troisi, R., et al. (2007) A rapid test of alloreactivity based on interleukin-2 mRNA expression might identify liver transplant recipients with donor-specific hyporesponsiveness. Transplant Proc 39, 2665–2667.

    Article  CAS  PubMed  Google Scholar 

  18. Ocmant, A., Michils, A., Schandene, L., et al. (2005) IL-4 and IL-13 mRNA real-time PCR quantification on whole blood to assess allergic response. Cytokine 31,375–381.

    Article  CAS  PubMed  Google Scholar 

  19. Chomczynski, P., Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162,156–159.

    Article  CAS  PubMed  Google Scholar 

  20. Rozen, S., Skaletsky, H. (2000) WWW for general users and for biologist programmers, in Bioinformatics Methods and Protocols: Methods in Molecular Biology (Krawetz, S. and Misener, S. ed.), Humana, Totowa, NJ,pp. 365–386.

    Google Scholar 

  21. Dheda, K., Huggett, J. F., Bustin, S. A., et al. (2004) Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques 37, 112–119.

    CAS  PubMed  Google Scholar 

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Stordeur, P. (2009). Monitoring the Immune Response Using Real-Time PCR. In: Bugert, P. (eds) DNA and RNA Profiling in Human Blood. METHODS IN MOLECULAR BIOLOGY™, vol 496. Humana Press. https://doi.org/10.1007/978-1-59745-553-4_22

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  • DOI: https://doi.org/10.1007/978-1-59745-553-4_22

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-934115-93-0

  • Online ISBN: 978-1-59745-553-4

  • eBook Packages: Springer Protocols

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