Abstract
Novel biomarkers are sought after by mining DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues. Such tissues offer the great advantage of often having complete clinical data (including survival), as well as the tissues are amenable for laser microdissection targeting specific tissue areas. Downstream analysis of such DNA includes mutational screens and methylation profiling. Screening for mutations by sequencing requires a significant amount of DNA for PCR and cycle sequencing. This is self-inhibitory if the gene screened has a large number of exons. Profiling DNA methylation using the MethyLight technology circumvents this problem and allows for the mining of several biomarkers from DNA extracted from a single microscope slide of the tissue of interest. We describe in this chapter a detailed protocol for MethyLight and its use in the determination of CpG Island Methylator Phenotype status in FFPE colorectal cancer samples.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Al-Mulla, F. (2007) Utilization of microarray platforms in clinical practice: an insight on the preparation and amplification of nucleic acids from frozen and fixed tissues. Methods Mol Biol. 382, 115–136.
Gagnon, J. F., Sanschagrin, F., Jacob, S., Tremblay, A. A., Provencher, L., Robert, J., et al. (2009) Quantitative DNA methylation analysis of laser capture microdissected formalin-fixed and paraffin-embedded tissues. Exp Mol Pathol. 8, 8.
Dietrich, D., Lesche, R., Tetzner, R., Krispin, M., Dietrich, J., Haedicke, W., et al. (2009) Analysis of DNA methylation of multiple genes in microdissected cells from formalin-fixed and paraffin-embedded tissues. J Histochem Cytochem. 57, 477–489.
Clark, S. J., Harrison, J., Paul, C. L., and Frommer, M. (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22, 2990–2997.
Eads, C. A. and Laird, P. W. (2002) Combined bisulfite restriction analysis (COBRA). Methods Mol Biol. 200, 71–85.
Herman, J. G., Graff, J. R., Myohanen, S., Nelkin, B. D., and Baylin, S. B. (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. 93, 9821–9826.
Eads, C. A., Danenberg, K. D., Kawakami, K., Saltz, L. B., Blake, C., Shibata, D., et al. (2000) MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res. 28, E32.
Issa, J. P. (2004) CpG island methylator phenotype in cancer. Nat Rev Cancer. 4, 988–993.
Widschwendter, M., Siegmund, K. D., Muller, H. M., Fiegl, H., Marth, C., Muller-Holzner, E., et al. (2004) Association of breast cancer DNA methylation profiles with hormone receptor status and response to tamoxifen. Cancer Res. 64, 3807–3813.
Weisenberger, D. J., Siegmund, K. D., Campan, M., Young, J., Long, T. I., Faasse, M. A., et al. (2006) CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet. 38, 787–793.
Ogino, S., Brahmandam, M., Kawasaki, T., Kirkner, G. J., Loda, M., and Fuchs, C. S. (2006) Epigenetic profiling of synchronous colorectal neoplasias by quantitative DNA methylation analysis. Mod Pathol. 19, 1083–1090.
Esteller, M. (2003) Relevance of DNA methylation in the management of cancer. Lancet Oncol. 4, 351–358.
Acknowledgments
This work is supported by Kuwait Foundation for the Advancement of Sciences grant number 2006-1302-07, Kuwait University Grant number MG02/08, and Research Core Facility (RCF) grant number GM 01/01and GM 01/05.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Dallol, A., Al-Ali, W., Al-Shaibani, A., Al-Mulla, F. (2011). Analysis of DNA Methylation in FFPE Tissues Using the MethyLight Technology. In: Al-Mulla, F. (eds) Formalin-Fixed Paraffin-Embedded Tissues. Methods in Molecular Biology, vol 724. Humana Press. https://doi.org/10.1007/978-1-61779-055-3_13
Download citation
DOI: https://doi.org/10.1007/978-1-61779-055-3_13
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-054-6
Online ISBN: 978-1-61779-055-3
eBook Packages: Springer Protocols