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DNA Methylation Protocols pp 49–58Cite as

Quantification of Global DNA Methylation Levels by Mass Spectrometry

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1708))

Abstract

Global DNA methylation was classically considered the relative percentage of 5-methylcysine (5mC) with respect to total cytosine (C). Early approaches were based on the use of high-performance separation technologies and UV detection. However, the recent development of protocols using mass spectrometry for the detection has increased sensibility and permitted the precise identification of peak compounds based on their molecular masses. This allows work to be conducted with much less genomic DNA starting material and also to quantify 5-hydroxymethyl-cytosine (5hmC), a recently identified form of methylated cytosine that could play an important role in active DNA demethylation. Here, we describe the protocol that we currently use in our laboratory to analyze 5mC and 5hmC by mass spectrometry. The protocol, which is based on the method originally developed by Le and colleagues using Ultra Performance Liquid Chromatography (UPLC) and mass spectrometry (triple Quadrupole (QqQ)) detection, allows for the rapid and accurate quantification of relative global 5mC and 5hmC levels starting from just 1 μg of genomic DNA, which allows for the rapid and accurate quantification of relative global 5mC and 5hmC levels.

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Acknowledgments

We thank Ronnie Lendrum for editorial assistance. This work has been financially supported by the following: Fondo de Investigaciones Sanitarias FIS/FEDER (PI11/01728 to AF.F. and PI12/01080 to M.F.F.); the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (Miguel Servet contract: CP11/00131 to A.F.F.); the Spanish National Research Council (CSIC; 200820I172 to M.F.F.); Fundación Ramón Areces (to M.F.F). The IUOPA is supported by the Obra Social Cajastur, Spain.

Author information

Author notes

  1. Agustin F. Fernandez and Luis Valledor contributed equally to this work.

Authors and Affiliations

  1. Cancer Epigenetics Laboratory, Institute of Oncology of Asturias (IUOPA), HUCA, Universidad de Oviedo, Oviedo, Spain

    Agustin F. Fernandez & Mario F. Fraga

  2. Area de Fisiología Vegetal, Dpto. Biología de Organismos y Sistemas, Universidad de Oviedo, Oviedo, Spain

    Luis Valledor & Maria Jesús Cañal

  3. Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Campus Universitario de Espinardo, Murcia, Spain

    Fernando Vallejo

  4. Department of Immunology and Oncology, National Center for Biotechnology, CNB-CSIC, Cantoblanco, 28049, Madrid, Spain

    Mario F. Fraga

  5. Instituto de Investigación Sanitaria del Principado de Asturias (IISPA), Hospital Universitario Central de Asturias (HUCA), Fundación para la Investigación Biosanitaria de Asturias (FINBA), Grupo de Epigenética del Cáncer y Nanomedicina, Avda. de Roma, s/n., 33011, Oviedo, Spain

    Agustin F. Fernandez & Mario F. Fraga

Authors
  1. Agustin F. Fernandez
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  2. Luis Valledor
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  3. Fernando Vallejo
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  4. Maria Jesús Cañal
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  5. Mario F. Fraga
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Corresponding author

Correspondence to Mario F. Fraga .

Editor information

Editors and Affiliations

  1. Laboratory for Epigenetics and Environment, Centre National de Recherche en Génomique Humaine, CEA—Institut de Biologie Francois Jacob, Evry, France

    Jörg Tost

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Fernandez, A.F., Valledor, L., Vallejo, F., Cañal, M.J., Fraga, M.F. (2018). Quantification of Global DNA Methylation Levels by Mass Spectrometry. In: Tost, J. (eds) DNA Methylation Protocols. Methods in Molecular Biology, vol 1708. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7481-8_3

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  • DOI: https://doi.org/10.1007/978-1-4939-7481-8_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7479-5

  • Online ISBN: 978-1-4939-7481-8

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