Abstract
Genome-wide sequence-specific methylation analysis has become a readily available and affordable procedure in epigenetic laboratories. Most of these procedures are based on immunoprecipitation, microarrays, or next generation deep bisulfite sequencing. However, most of these protocols are far from being quantitative. Moreover, abnormal or specific methylation patterns must always be further validated by quantitative sequence-specific methylation analysis. In this chapter, we describe a detailed and simplified protocol (using one universal HPLC gradient for all separations as well as one SNuPE annealing temperature for all primers) for the previously published SIRPH analysis, which is based on the single nucleotide primer extension combined with high-performance liquid chromatography. This method is highly accurate, reproducible, quantitative, and suitable for analysis of as little as 50 ng of PCR product derived from limited starting materials.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Lister, R., Pelizzola, M., Dowen, R.H., Hawkins, R.D., Hon, G., Tonti-Filippini, J., Nery, J.R., Lee, L., Ye, Z., Ngo, Q.M., Edsall, L., Antosiewicz-Bourget, J., Stewart, R., Ruotti, V., Millar, A.H., Thomson, J.A., Ren, B., Ecker, J.R. (2009) Human DNA methylomes at base resolution show widespread epigenomic differences. Nature. 462: 315–22.
Laurent, L., Wong, E., Li, G., Huynh, T., Tsirigos, A., Ong, C.T., Low, H.M., Kin Sung, K.W., Rigoutsos, I., Loring, J., Wei, C.L. (2010) Dynamic changes in the human methylome during differentiation. Genome Res. 20:320–31.
Kriaucionis, S., Heintz, N. (2009) The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science. 324:929–930.
Szwagierczak, A., Bultmann, S., Schmidt, C.S., Spada, F., Leonhardt, H. (2010) Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA. Nucleic Acids Res. 38:e181.
El-Maarri, O., Seoud, M., Coullin, P., Herbiniaux, U., Oldenburg, J., Rouleau, G., Slim, R. (2003) Maternal alleles acquiring paternal methylation patterns in biparental complete hydatidiform moles. Hum Mol Genet. 12:1405–13.
El-Maarri, O., Becker, T., Junen, J., Manzoor, S.S., Diaz-Lacava, A., Schwaab, R., Wienker, T., Oldenburg, J. (2007) Gender specific differences in levels of DNA methylation at selected loci from human total blood: a tendency toward higher methylation levels in males. Hum Genet. 122:505–14.
El-Maarri, O., Kareta, M.S., Mikeska, T., Becker, T., Diaz-Lacava, A., Junen, J., Nüsgen, N., Behne, F., Wienker, T., Waha, A., Oldenburg, J., Chédin, F. (2009) A systematic search for DNA methyltransferase polymorphisms reveals a rare DNMT3L variant associated with subtelomeric hypomethylation. Hum Mol Genet. 18:1755–68.
Fuke, C., Shimabukuro, M., Petronis, A., Sugimoto, J., Oda, T., Miura, K., Miyazaki, T., Ogura, C., Okazaki, Y., Jinno, Y. (2004) Age related changes in 5-methylcytosine content in human peripheral leukocytes and placentas: an HPLC-based study. Ann Hum Genet. 68:196–204.
Bollati, V., Baccarelli, A., Hou, L., Bonzini, M., Fustinoni, S., Cavallo, D., Byun, H.M., Jiang, J., Marinelli, B., Pesatori, A.C., Bertazzi, P.A., Yang, A.S. (2007) Changes in DNA methylation patterns in subjects exposed to low-dose benzene. Cancer Res. 67:876–80.
Wolff, G.L., Kodell, R.L., Moore, S.R., Cooney, C.A. (1998) Maternal epigenetics and methyl supplements affect agouti gene expression in Avy/a mice. FASEB J. 12:949–57.
Ramsahoye, B.H. (2002) Measurement of genome wide DNA methylation by reversed-phase high-performance liquid chromatography. Methods. 27:156–61.
Fraga, M.F., Uriol, E., Borja, Diego, L., Berdasco, M., Esteller, M., Cañal, M.J., Rodríguez, R. (2002) High-performance capillary electrophoretic method for the quantification of 5-methyl 2′-deoxycytidine in genomic DNA: application to plant, animal and human cancer tissues. Electrophoresis. 23:1677–81.
Stach, D., Schmitz, O.J., Stilgenbauer, S., Benner, A., Döhner, H., Wiessler, M., Lyko, F. (2003) Capillary electrophoretic analysis of genomic DNA methylation levels. Nucleic Acids Res. 31:e2.
Karimi, M., Johansson, S., Stach, D., Corcoran, M., Grandér, D., Schalling, M., Bakalkin, G., Lyko, F., Larsson, C., Ekström. (2006) LUMA (LUminometric Methylation Assay)–a high throughput method to the analysis of genomic DNA methylation. TJ. Exp Cell Res. 312:1989–95.
Weber, M., Davies, J.J., Wittig, D., Oakeley, E.J., Haase, M., Lam, W.L., Schübeler, D. (2005) Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet. 37:853–62.
Tost, J., Dunker, J., Gut, I.G. (2003) Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing. Biotechniques. 35:152–6.
El-Maarri, O., Herbiniaux, U., Walter, J., Oldenburg, J. (2002) A rapid, quantitative, non-radioactive bisulfite-SNuPE- IP RP HPLC assay for methylation analysis at specific CpG sites. Nucleic Acids Res. 30:e25.
El-Maarri, O. (2004) SIRPH analysis: SNuPE with IP-RP-HPLC for quantitative measurements of DNA methylation at specific CpG sites. Methods Mol Biol. 287:195–205.
Syvanen, A.C. (1999) From gels to chips: “minisequencing” primer extension for analysis of point mutations and single nucleotide polymorphisms. Hum Mutat. 13:1–10.
Gonzalgo, M.L., Jones, P.A. (1997) Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res. 25:2529–2531.
Tost, J., Schatz, P., Schuster, M., Berlin, K. Gut, I.G. (2003) Analysis and accurate quntification by MALDI mass spectrometry. Nucleic Acids Res. 31:e50.
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
Singer, H., Nüsgen, N., El-Maarri, O. (2011). SIRPH: An HPLC-Based SNuPE for Quantitative Methylation Measurement at Specific CpG Sites. In: Tollefsbol, T. (eds) Epigenetics Protocols. Methods in Molecular Biology, vol 791. Humana Press. https://doi.org/10.1007/978-1-61779-316-5_7
Download citation
DOI: https://doi.org/10.1007/978-1-61779-316-5_7
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-315-8
Online ISBN: 978-1-61779-316-5
eBook Packages: Springer Protocols