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 analyzes the methylation status of more than 450.000 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 preliminary HumanMethylation27 content. The HumanMethylation27 is successfully used at GPCF since end of 2008. The requirements for your genomic DNA samples have remained the same. (see below)

We recently established 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.

YOU supply

• 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

WE perform

• 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

• 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

Pricing per DNA sample is provided on our pricelist. For further details concerning methylation analysis, please feel free to contact Matthias Schick.