Genome-wide Methylation Analysis
Epigenetics describes the study of heritable changes in gene function that occur without a change in the nuclear DNA sequence. A major epigenetic mechanism in higher eukaryotes is DNA methylation. It involves the covalent addition of a methyl group to the 5'-carbon of cytosine usually in a CpG dinucleotide.
Many, if not all, human tumors exhibit an altered methylation signature. This aberrant methylation pattern is often characterized by global hypomethylation and locus specific hypermethylation, which often occurs in promoter regions of tumorsuppressor genes such as p16 or RASSF1 (Esteller, 2007). Most frequently DNA methylation is seen in promotor regions of genes, often extending through the first exon and into the first intron (Zilberman, 2007). In addition, transposons are often methylated heavily.
Methylation patterns are maintained during replication (semi-conservative duplication of DNA) by DNA Methyltransferase 1 (DNMT1). De novo methylation of CpG sites is carried out by DNMT3A and DNMT3B. The methylation patterns allow distinctions according to cell types, age, or cancer (vs. normal).
At GPCF microarray unit, we provide full service for the analysis of DNA methylation on a genome-wide level in human samples (genomic DNA derived from fresh frozen or FFPE material) making use of the well established Infinium technology of Illumina.
The HumanMethylation450 beadchip (launched January 2011) analyzes the methylation status of 485.577 CpG dinucleotides in parallel. This allows a genome-wide and cost effective insight into the human methylome at single-nucleotide resolution.
The interrogated sites have been selected to represent the following content:
- coverage of all designable RefSeq genes (also with lacking CpG islands), including promoter, 5', and 3' regions
- CpG islands and shores
- CpG sites outside of CpG islands
- Non-CpG methylated sites identified in human stem cells
- tumor relevant differentially methylated sites
- CpG islands outside of coding regions
- miRNA promoter regions
- Disease-associated regions identified through GWAS
To provide basic comparability the HumanMethylation450 contains 90% of the preceding HumanMethylation27 content. The HumanMethylation27 is successfully used at GPCF since end of 2008 and has been discontinued by Illumina since June 2012 as in the meantime all user have switched to the new format. The requirements for your genomic DNA samples have remained the same. (see below)
Since 2012 we offer a modified version of the Infinium Methylation protocol, which allows methylation studies on FFPE samples. The quality of such material strongly varies depending on applied FFPE protocol and storage period. Therefore, we perform an initial quality control with all incoming samples using fluorescence based quantification followed by quantitative Real-Time PCR. Only samples passing this additional QC will be bisulfite converted and processed in a special restore step followed by the standard hybridisation workflow. Due to this additional effort an extra FFPE fee per sample will be charged.
- Completion of sample sheet
- 12 DNA samples (or multiple thereof) are required in each request. Experiments containing more than 24 samples should be announced 4-6 weeks in advance. This is due to the fact that our stock is limited and the delivery period is minimum 4 weeks.
- DNA from fresh frozen material: >1,5 µg DNA with av. fragment size >3kb (free of organic solvents) and 50ng/µl - 200ng/µl concentration
- DNA from FFPE material: > 500ng and a minimum concentration of 25ng/µl
- QC of DNA sample (applied methods depend on the DNA's origin)
- Bisulfite treatment (incl. QC)
- Restore procedure (only for FFPE samples)
- Infinium assay (technical reproducibility >0,98, PCR free assay)
- Basic data analysis
- Raw data
- Methylation information on all CpG sites tested (in MS Excel 2007 compatible format)
- Medium term storage of all data for at least 12 months
- Discussion on the results
Citing our service
Please cite our service in the acknowledgements part of your paper in case we've just performed the standard service. The following statement has been used in the past.:
"We thank the microarray unit of the DKFZ Genomics and Proteomics Core Facility for providing the Illumina Human Methylation arrays and related services."
Find below a selection of publications making use of data generated at the microarray unit of the GPCF using HumanMethylation450 or HumanMethylation27.
Hovestadt et al.
Acta Neuropathol. 2013 Jun;125(6):913-916
Robust molecular subgrouping and copy-number profiling of medulloblastoma from small amounts of archival tumour material using high-density DNA methylation arrays.
Lambert et al.
Acta Neuropathol. 2013 May 10
Differential expression and methylation of brain developmental genes define location-specific subsets of pilocytic astrocytoma.
Sturm et al.
Cancer Cell. 2012 Oct 16;22(4):425-37
Hotspot Mutations in H3F3A and IDH1 Define Distinct Epigenetic and Biological Subgroups of Glioblastoma.
Koch et al.
Genome Res. 2013 Feb;23(2):248-59
Pluripotent stem cells escape from senescence-associated DNA methylation changes.
Bocker et al.
Nat Commun. 2012 May 8;3:818.
Hydroxylation of 5-methylcytosine by TET2 maintains the active state of the mammalian HOXA cluster.
Hagemann et al.
PLoS One. 2012;7(5):e36125. Epub 2012 May 1.
Antiproliferative effects of DNA methyltransferase 3B depletion are not associated with DNA demethylation.
Breitling et al.
Am J Hum Genet. 2011 Apr 8;88(4):450-7. Epub 2011 Mar 31.
Tobacco-smoking-related differential DNA methylation: 27K discovery and replication.
Bocker et al.
Blood. 2011 May 12;117(19):e182-9. Epub 2011 Mar 22.
Genome-wide promoter DNA methylation dynamics of human hematopoietic progenitor cells during differentiation and aging.