Assessment of Global DNA Methylation in Stem Cells with Reverse-Phase High Performance Liquid Chromatography

Abstract

Objective: We tested the possibility of the use of reverse-phase high performance liquid chromatography (RP-HPLC), a sensitive, simple and cost effective technique, for evaluation of global DNA methylation alteration in stem cells. Methods: We detected genomic methylation in four cell lines of mouse embryonic stem cells and in the cell at various developmental stages, including mouse embryonic fibroblast (n=5) and mouse tissues (n=9), using RP-HPLC. The samples were extracted for single nucleosides and investigated for the concentrations of deoxy-cytidine (dC), deoxy-guanosine (dG) and deoxymethyl-cytidine (5-dmC) using RP-HPLC. The methylation levels were obtained by a ratio of the entire genomic 5m-C to dC. Results: Our results demonstrated that the RP-HPLC technique can be used for analysis of global methylation on the stem cell genome. It can display the discrepancy of methylation levels among the cells at different degrees of cell differentiation. Conclusion: We present the evidence that the RP-HPLC technique can be used for the entire-genomic methylation detection in stem cells and should be further developed as a strategy for monitoring the global DNA methylation of stem cells in advanced research.

Keywords

• DNA methylation
• epigenetics
• methyl cytidine
• reverses phase-HPLC
• stem cells