Methods used for methylation analyses
Table of Contents
Currently, two approaches are utilized in the division to analyze epigenetic alterations in cancer development: A genome-wide approach to identify novel hypermethylated CpG islands, and a quantitative candidate-gene approach.
Genome wide methylation analysis
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 and two-dimensional gel electrophoresis (Plass et al., Oncogene 1999; Costello et al., Nat. Genet. 2000).
Methodologic development during the past years has allowed to replace RLGS by novel detection technology based on methylation-specific DNA array analysis and next-generation sequencing after methyl-CpG immunoprecipitation (MCIp)(Gebhard et al., Cancer Res. 2006).
Method description: Methylome analysis
High-throughput quantitative candidate gene methylation analysis
For the candidate gene approach, techniques established in the division include the Bio-COBRA assay (Brena et al., Nat. Protoc. 2006) and the EpiTYPER - MassARRAY system from Sequenom Inc.
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, Bennett et al., Cancer Res. 2008).
Method description: EpiTYPER - MassARRAY
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), siRNA 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.