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High-Resolution Quantitative Methylation Analysis of MicroRNA Genes Using Pyrosequencing™

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Metastasis Research Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 878))

Abstract

MicroRNA (miRNA) genes have been shown to perform a crucial role in breast cancer metastasis. The epigenetic inactivation of such microRNA genes, as a result of aberrant DNA methylation, is frequently found in human tumours including those of the breast, and this is an area of considerable research activity.

Pyrosequencing™ is a new quantitative method for the assessment of DNA methylation, with single CpG site resolution. Pyrosequencing™ can easily be performed in a 96-well-plate format with a cost-effective medium-sized throughput.

This chapter provides a general outline of DNA methylation analysis, a detailed protocol of the Pyrosequencing™ procedure, and guidelines for the design of new assays. The strengths and limitations of this approach are discussed throughout the chapter.

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Acknowledgements

The authors would like to thank Britta Hasemeier for expert assistance in preparing the figures and Dr. Florian Puls for critically reading the manuscript.

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Authors and Affiliations

  1. Institute of Pathology, Medizinische Hochschule Hannover, Hannover, Germany

    Ulrich Lehmann, Cord Albat & Hans Kreipe

Authors
  1. Ulrich Lehmann
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  2. Cord Albat
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  3. Hans Kreipe
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Corresponding author

Correspondence to Ulrich Lehmann .

Editor information

Editors and Affiliations

  1. School of Life Sciences, Molecular and Applied Biosciences, University of Westminster, New Cavendish Street 115, London, W1W 6UW, United Kingdom

    Miriam Dwek

  2. School of Life Sciences, Oxford Brookes University, Gipsy Lane, Oxford, OX3 0BP, United Kingdom

    Susan A. Brooks

  3. Inst. Anatomie und Experimentelle, Morphologie, Universitätsklinikum Hamburg, Martinistr. 52, Hamburg, 20246, Germany

    Udo Schumacher

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Lehmann, U., Albat, C., Kreipe, H. (2012). High-Resolution Quantitative Methylation Analysis of MicroRNA Genes Using Pyrosequencing™. In: Dwek, M., Brooks, S., Schumacher, U. (eds) Metastasis Research Protocols. Methods in Molecular Biology, vol 878. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-854-2_15

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  • DOI: https://doi.org/10.1007/978-1-61779-854-2_15

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-853-5

  • Online ISBN: 978-1-61779-854-2

  • eBook Packages: Springer Protocols

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