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 HumanMethylationEPIC is the next generation of Infinium Methylation and builds upon the HumanMethylation450k (see below) with 93.3% of the original CpGs plus roughly additional 350.000 CpGs in enhancer regions:
- FANTOM5 enhancers
- ENCODE open chromatin and enhancers
- DNase hypersensitive sites
The MethylationEPIC array provides an unprecedented view of 866.895 CpGs in an 8 sample per chip format, utilizing the same chemistry and workflow as the original HumanMethylation450K BeadChip.
The HumanMethylation450 beadchip 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
- Differentially methylated sites identified in tumor versus normal CpG islands outside of coding regions
- miRNA promoter regions
- Disease-associated regions identified through GWAS
Please note: The 450k array is still available for a limited time to finish open projects and will be replaced by the Methylation EPIC!
Methylation studies on FFPE samples are enabled with a modified version of the Infinium Methylation protocol. 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
- DNA Sample Concentration and Volume Requirements:
|Chip-Format||12 samples||8 samples|
|Total DNA/sample||>1000 ng||>1000 ng|
|Adjust sample volume to||40 Âµl||40 Âµl|
|Total DNA/sample||>500 ng||>500 ng|
|Adjust sample volume to||40 Âµl||40 Âµl|
For lower DNA amounts please inquire.
- 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
WE provide (see example expression data)
- 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 HumanMethylationEPIC.
Capper D, et al.
DNA methylation-based classification of central nervous system tumours.
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.