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Epigenetic Methodologies for the Study of Celiac Disease

  • Protocol
Book cover Celiac Disease

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1326))

Abstract

Epigenetic regulation of gene expression is an important event for normal cellular homeostasis. Gene expression may be “switched” on or “turned” off via epigenetic means through adjustments in the architecture of DNA. These structural alterations result from histone posttranslation modifications such as acetylation and methylation on key arginine and lysine residues, or by alterations to DNA methylation. Other known epigenetic mechanisms invoke histone variant exchange or utilize noncoding RNAs (lncRNA/miRNA). Drugs which can target the epigenetic regulatory machinery are currently undergoing clinical trials in a wide variety of autoimmune diseases and cancer.

Here we describe RNA isolation and the subsequent Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) methods, post-epigenetic drug treatment, to identify genes, which may be responsive to such epigenetic targeting agents. In addition, we depict a chromatin immunoprecipitation (ChIP) assay to determine the association between chromatin transcription markers and DNA following pretreatment of cell cultures with a histone deacetylase inhibitor (HDi). This assay allows us to determine whether treatment with an HDi dynamically remodels the promoter region of genes, as judged by the differences in the PCR product between our treated and untreated samples. Finally we describe two commonly used methodologies for analyzing DNA methylation. The first, methylation-sensitive high resolution melt analysis (MS-HRM) is used for methylation screening of regions of interest, to identify potential epigenetic “hotspots.” The second, quantitative methylation specific PCR (qMSP) is best applied when these hotspots are known, and offers a high-throughput, highly sensitive means of quantifying methylation at specific CpG dinucleotides.

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Acknowledgements

The authors gratefully acknowledge the contribution of Alexandra Tuzova in the design of Fig. 1.

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Authors and Affiliations

  1. Prostate Molecular Oncology, Institute of Molecular Medicine, Trinity College Dublin, Dublin, Ireland

    Antoinette S. Perry

  2. Department of Clinical Medicine, Trinity College Dublin, Dublin, Ireland

    Anne-Marie Baird & Steven G. Gray

  3. Thoracic Oncology Research Group, Institute of Molecular Medicine, Trinity Centre for Health Science, St. James’s Hospital, Dublin 8, Ireland

    Anne-Marie Baird & Steven G. Gray

  4. Cancer & Ageing Research Program, Queensland University of Technology, Brisbane, QLD, Australia

    Anne-Marie Baird

  5. HOPE Directorate, St. James’s Hospital, Dublin, Ireland

    Steven G. Gray

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  1. Antoinette S. Perry
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  2. Anne-Marie Baird
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  3. Steven G. Gray
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Correspondence to Steven G. Gray .

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Editors and Affiliations

  1. Trinity Centre for Health Sciences, Trinity College Dublin Trinity Centre for Health Sciences, Dublin, Ireland

    Anthony W. Ryan

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Perry, A.S., Baird, AM., Gray, S.G. (2015). Epigenetic Methodologies for the Study of Celiac Disease. In: Ryan, A. (eds) Celiac Disease. Methods in Molecular Biology, vol 1326. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2839-2_13

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  • DOI: https://doi.org/10.1007/978-1-4939-2839-2_13

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2838-5

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