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Studying the Replication History of Human B Lymphocytes by Real-Time Quantitative (RQ)-PCR

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Lymphoma

Part of the book series: Methods in Molecular Biology ((MIMB,volume 971))

Abstract

The cells of the adaptive immune system, B and T lymphocytes, each generate a unique antigen receptor through V(D)J recombination of their immunoglobulin (Ig) and T cell receptor (TCR) loci, respectively. Such rearrangements join coding elements to form a coding joint and delete the intervening DNA as circular excision products containing the signal joint. These excision circles are stable structures that cannot replicate and have no function in the cell. Since the coding joint in the genome is replicated with each cell division, the ratio between coding joints and signal joints in a population of B cells can be used as a measure for proliferation. This chapter describes a real-time quantitative (RQ-)PCR-based approach to quantify proliferation through calculating the ratio between coding joints and signal joints of the frequently occurring intronRSS–Kde rearrangements in the IGK light chain locus. The approach is useful to study basic B-cell biology as well as abnormal proliferation in human diseases.

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Abbreviations

BCR:

B-cell antigen receptor

D:

Diversity

Ig:

Immunoglobulin

IgH:

Ig heavy chain

IgÎş:

Ig kappa light chain

Igλ:

Ig lambda light chain

J:

Joining

Kde:

Kappa-deleting element

KREC:

Kappa-deleting recombination excision circle

RAG:

Recombination activating gene

RQ-PCR:

Real-time quantitative PCR

RSS:

Recombination signal sequence

V:

Variable

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Acknowledgments

MAB is supported by a Fellowship from the Ter Meulen Fund–Royal Netherlands Academy of Arts and Sciences. MCvZ is supported by an Erasmus University Rotterdam (EUR)-Fellowship, an Erasmus MC Fellowship, and by Veni grant 916.110.90 from ZonMW/NWO and by grant 689 of the Sophia Children’s Hospital Fund.

Conflict of Interest

JJMvD is inventor of the KREC assay, which has been patented (PCT/NL 2005/000761; priority date 25 Oct 2004) and licensed to InVivoScribe Technologies, San Diego, CA; revenues of the patent go to Erasmus MC. The other authors have declared that no conflict of interest exists.

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Authors and Affiliations

  1. Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands

    Menno C. van Zelm, Magdalena A. Berkowska & Jacques J. M. van Dongen

Authors
  1. Menno C. van Zelm
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  2. Magdalena A. Berkowska
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  3. Jacques J. M. van Dongen
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Corresponding author

Correspondence to Menno C. van Zelm .

Editor information

Editors and Affiliations

  1. Medical School, Institute of Cell Biology (Cancer Resear, University of Duisburg-Essen, Virchowstr. 173, Essen, 45122, Germany

    Ralf KĂĽppers

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van Zelm, M.C., Berkowska, M.A., van Dongen, J.J.M. (2013). Studying the Replication History of Human B Lymphocytes by Real-Time Quantitative (RQ)-PCR. In: KĂĽppers, R. (eds) Lymphoma. Methods in Molecular Biology, vol 971. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-269-8_6

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  • DOI: https://doi.org/10.1007/978-1-62703-269-8_6

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-268-1

  • Online ISBN: 978-1-62703-269-8

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