Abstract

Alterations of normal gene expression patterns are a hallmark of human cancers. It is now clear that the dysregulation of epigenetic modifications of the DNA and surrounding histones contributes to aberrant gene silencing, thus being major participants not only in the progression but also the initiation of the disease phenotype. The best-studied epigenetic modification is DNA methylation, which converts cytosine to 5-methylcytosine. Aberrant hypermethylation of the promoter is frequently observed in cancer and is generally associated with gene silencing. Currently, 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. In the life sciences, microfluidics usually entails the scaling down of at least one application, such as electrophoresis, to chip format, which often results in increased efficiency and reliability. Bio-COBRA provides a platform for the rapid and quantitative assessment of DNA methylation patterns in large sample sets. Its sensitivity and reproducibility also makes it a tool for the analysis of DNA methylation in clinical samples. The Bio-COBRA assay can be performed on 12 samples in less than 1 h. If the protocol is started at the DNA isolation step, however, approximately 48 h would be required to complete the entire procedure.