Bio-COBRA: Absolute Quantification of DNA Methylation in Electrofluidics Chips
DNA methylation is the best-studied epigenetic modification, and in mammals it describes the conversion of cytosine to 5-methylcytosine in the context of CpG dinucleotides. In recent years, it has become evident that epigenetic mechanisms are severely disrupted in human neoplasia, and evidence suggests that alterations of DNA methylation patterns may be an integral mechanism in the etiology of other diseases such as bipolar disorder and schizophrenia. The main effect of altered DNA methylation is the disruption of normal patterns of gene expression through genomic instability and hypermethylation of CpG islands, which together could lead to uncontrolled cell proliferation. DNA methylation can be reversed through pharmacological intervention via the systemic administration of DNA methylation inhibitors. Thus, the ability to accurately quantify DNA methylation levels in genomic sequences is a prerequisite to assess not only treatment efficacy, but also the effect of the DNA methylation inhibitors on bystander tissues. Several methods are currently available for the analysis of DNA methylation. Nonetheless, accurate and reproducible quantification of DNA methylation remains challenging. Here, we describe Bio-COBRA, a modified protocol for combined bisulfite restriction analysis (COBRA) that incorporates an electrophoresis step in microfluidics chips. Microfluidics technology involves the handling of small amounts of liquid in miniaturized systems. Bio-COBRA provides a platform for the rapid and quantitative assessment of DNA methylation patterns in large sample sets. Its sensitivity and reproducibility also make it an excellent tool for the analysis of DNA methylation in clinical samples.
KeywordsBio-COBRA electrofluidics chips quantification DNA methylation DNA methylation inhibitor dynamic range, 2100 Bioanalyzer
We would like to thank Herbert Auer and Dr. Karl Kornacker, both of whom were involved in the development and testing of this protocol.