Methods used for methylation analyses
Overview
For the detection of aberrant DNA methylation, we utilize a set of genome-wide methylation profiling methods. Differential methylation is validated by quantitative, sequence-specific high-throughput EpiTyper MassARRAY technology based on MALDI-TOF mass spectroscopy. Validated findings are then confirmed in additional sample sets with clinical follow-up that allow to identify methylation-based biomarkers for cancer detection or prediction of prognosis. Interesting findings on aberrant methylation and alterations in gene expression are then followed by functional analyses using gain- and loss-of-function strategies.
Analyzing the epigenome
Modern epigenomics includes the quantitative determination of genome-wide DNA methylation and non-coding RNA expression, and analyzes structure and modifications of the chromatin. Functional studies of genetic and epigenetic regulation complement these profiling approaches.
The Plass group has gained international recognition for the establishment and improvement of the method of 'Restriction Landmark Genomic Scanning' (RLGS), which is a combination of methylation-sensitive restriction digestion of genomic DNA and two-dimensional gel electrophoresis (Plass et al., Oncogene 1999; Costello et al., Nat. Genet. 2000).
In order to keep pace with recent methodologic developments, we successively replaced RLGS by array- and next generation sequencing-based detection technologies. 'Methyl-CpG immunoprecipitation' enables array- or sequencing-based analysis of enriched methylated DNA (MCIp-CpG island array, MCIp-Seq) (Gebhard et al., Cancer Res. 2006). The Illumina 450k bead chip array platform provides quantitative methylation data for about 450.000 preselected CpG sites across the human genome. We have established 'Tagmentation-based whole genome bisulfite sequencing' (TB-WGBS)(Adey & Shendure, Genome Res. 2012; Lu et al., Epigenomics 2015) to interrogate the quantitative methylation states of all CpGs in human, mouse or other genome from less than 1,000 cells (Wang et al., Nat. Protoc. 2013). In addition, we apply the 'Assay for Transposase-Accessible Chromatin' (ATAC) (Buenrostro et al., Nat. Meth. 2013) and chromatin immunoprecipitation (ChIP) to analyze chromatin structure and modifications. To relate epigenomic alterations with expression changes, next generation-based transcriptome analysis is used to quantitatively analyze expression of mRNA, micro RNA and long-non coding RNA.
High-throughput quantitative methylation analysis
For quantitative methylation analysis we routinely use the EpiTyper - MassARRAY system from Sequenom Inc.
Mass spectrum displaying "methylated" and "unmethylated" peaks for all analyzed CpG units within one amplicon situated in a CpG-rich region of the ZAP70 gene.
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The MassARRAY system is ideal for discovery of DNA methylation, for discrimination between methylated and non-methylated samples, and for quantifying methylation levels of DNA in a high-throughput manner (Raval et al., Cell 2007, Claus et al., JCO 2012).
Functional analyses
Once a novel candidate gene has been identified, its functional role in carcinogenesis is investigated in vitro and in vivo.
For functional analyses, methods for quantitative real time PCR analyses, various blotting and hybridization techniques (FISH, Southern, Northern, Western blotting), si- and shRNA technology, cloning techniques, multiple luciferase-based reporter systems, and flow cytometry for apoptosis and cell cycle analysis are established. Demethylating agents for comparative mechanistic analyses are available.