Skip to main content
SpringerLink
Log in

Laser Capture Microdissection as a Tool to Study the Mucosal Immune Response in Celiac Disease

  • Protocol
  • First Online:
Laser Capture Microdissection

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1723))

Abstract

Laser capture microdissection (LCM) is a powerful tool for selection and isolation of single cells or compartments from complex primary tissues to perform molecular analyses. Celiac disease is a genetic autoimmune disorder where the ingestion of gluten leads to damage in the small intestine. Increased intraepithelial lymphocytes and the presence of the lamina propria inflammatory infiltrate of the duodenal mucosa is a common part of the disease. These cells promote inflammatory processes through the release of cytokines. Here, we describe the use of LCM and real-time quantitative PCR (RT-qPCR) to analyze cytokine profile information in distinct duodenal mucosa tissue compartments of celiac patients.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Datta S, Malhotra L, Dickerson R et al (2015) Laser capture microdissection: big data from small samples. Histol Histopathol 30:1255–1269

    CAS  Google Scholar 

  2. Blatt R, Srinivasan S (2008) Defining disease with laser precision: laser capture microdissection in gastroenterology. Gastroenterology 135:364–369

    Article  Google Scholar 

  3. Mazzarella G (2015) Effector and suppressor T cells in celiac disease. World J Gastroenterol 21:7349–7356

    Article  CAS  Google Scholar 

  4. Nilsen EM, Jahnsen FL, Lundin KE et al (1998) Gluten induces an intestinal cytokine response strongly dominated by interferon gamma in patients with celiac disease. Gastroenterology 115:551–563

    Article  CAS  Google Scholar 

  5. Troncone R, Gianfrani C, Mazzarella G et al (1998) Majority of gliadin-specific T-cell clones from celiac small intestinal mucosa produce interferon-gamma and interleukin-4. Dig Dis Sci 43:156–161

    Article  CAS  Google Scholar 

  6. Lahat N, Shapiro S, Karban A et al (1999) Cytokine profile in coeliac disease. Scand J Immunol 49:441–446

    Article  CAS  Google Scholar 

  7. Forsberg G, Hernell O, Melgar S et al (2002) Paradoxical coexpression of proinflamatory and down-regulatory cytokines in intestinal T cells in childhood celiac disease. Gastroenterology 123:667–678

    Article  CAS  Google Scholar 

  8. Di Sabatino A, Ciccocioppo R, Cupelli F et al (2006) Epithelium derived interleukin 15 regulates intraepithelial lymphocyte Th1 cytokine production, cytotoxicity, and survival in coeliac disease. Gut 55:469–477

    Article  Google Scholar 

  9. Fosberg G, Hernell O, Hammarstro¨m S et al (2007) Concomitant increase of IL-10 and pro-inflammatory cytokines in intraepithelial lymphocyte subsets in celiac disease. Int Immunol 19:993–1001

    Article  Google Scholar 

  10. Simone NL, Bonner RF, Gillespie Emmert-Buck MR et al (1998) Laser-capture microdissection: opening the microscopic frontier to molecular analysis. Trends Genet 14:272–276

    Article  CAS  Google Scholar 

  11. Curran S, McKay JA, McLeod HL et al (2000) Laser capture microscopy. Mol Pathol 53:64–68

    Article  CAS  Google Scholar 

  12. Lawrie LC, Curran S (2005) Laser-capture microdissection and colorectal cancer proteomics. Methods Mol Biol 293:245–253

    CAS  Google Scholar 

  13. Kerman IA, Buck BJ, Evans SJ et al (2006) Combining laser capture microdissection with quantitative real-time PCR: effects of tissue manipulation on RNA quality and gene expression. J Neurosci Methods 15(153):71–85

    Article  Google Scholar 

  14. Erickson HS, Albert PS, Gillespie JW et al (2009) Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples. Nat Protoc 4:902–922

    Article  CAS  Google Scholar 

  15. Goldsworthy SM, Stockton PS, Trempus CS et al (1999) Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue. Mol Carcinog 25(2):86–91

    Article  CAS  Google Scholar 

  16. Iacomino G, Marano A, Stillitano I et al (2016) Celiac disease: role of intestinal compartments in the mucosal immune response. Mol Cell Biochem 411:341–349

    Article  CAS  Google Scholar 

  17. Fleige S, Walf V, Huch S et al (2006) Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR. Biotechnol Lett 28:1601–1613

    Article  CAS  Google Scholar 

  18. Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622

    Article  CAS  Google Scholar 

  19. 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

    Article  Google Scholar 

  20. Heid CA, Stevens J, Livak KJ et al (1996) Real time quantitative PCR. Genome Res 6:986–994

    Article  CAS  Google Scholar 

  21. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C (T) method. Nat Protoc 3:1101–1108

    Article  CAS  Google Scholar 

  22. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors are grateful to Eng. Clemente Meccariello for his technical help.

Author information

Author notes

  1. Giuseppe Iacomino and Vera Rotondi Aufiero contributed equally to this work.

Authors and Affiliations

  1. Institute of Food Sciences—CNR, Avellino, Italy

    Giuseppe Iacomino, Vera Rotondi Aufiero, Pasquale Marena, Antonella Venezia & Giuseppe Mazzarella

  2. Department of Translational Medical Science, University of Naples “Federico II”, Naples, Italy

    Riccardo Troncone & Salvatore Auricchio

  3. European Laboratory for the Investigation of Food-Induced Disease, University of Naples “Federico II”, Naples, Italy

    Riccardo Troncone, Salvatore Auricchio & Giuseppe Mazzarella

Authors
  1. Giuseppe Iacomino
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vera Rotondi Aufiero
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pasquale Marena
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Antonella Venezia
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Riccardo Troncone
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Salvatore Auricchio
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Giuseppe Mazzarella
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Giuseppe Mazzarella .

Editor information

Editors and Affiliations

  1. School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Aberdeen, United Kingdom

    Graeme I. Murray

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Iacomino, G. et al. (2018). Laser Capture Microdissection as a Tool to Study the Mucosal Immune Response in Celiac Disease. In: Murray, G. (eds) Laser Capture Microdissection. Methods in Molecular Biology, vol 1723. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7558-7_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7558-7_7

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7557-0

  • Online ISBN: 978-1-4939-7558-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation