Quantifying Global DNA Methylation

Genomic DNA was isolated from hepatopancreas samples using the GenElute™ Mammalian Genomic DNA Miniprep Kit (Merck KGaA, Darmstadt, Germany) according to the manufacturer’s directions. The DNA purity was checked by UV absorption (260/280 nm, DS-11 Spectrophotometer, DeNovix, Wilmington, NC, USA) [7 (link)]. Total 5mC DNA level was quantified using the MethylFlash Global DNA Methylation (5mC) ELISA Easy Kit (EpiGentek, Farmingdale, NY, USA). This kit provides a fast and precise method to determine global DNA methylation changes, with a detection limit of 5 nanograms (ng) of fully methylated DNA. In addition, the use of such ELISA-based assays for serial measurements of 5mC can serve as a cost-effective and reliable alternative to commonly used methods for whole-genome DNA methylation quantitation, such as chromatography, radioactive filter-binding, or bisulfite sequencing-based methods [32 (link),33 (link)]. Optical density (OD) was measured at a wavelength of 450 nanometers (nm) with a Stat Fax 4200 microplate reader (Awareness Technology, Palm City, FL, USA). For absolute 5mC quantifications, a standard curve was created by graphing the corresponding ODs versus the log₁₀-transformed concentrations of the positive controls. The coefficients of determination (R²) for ELISA calibration curves were above 0.97; an example is shown in Figure 1.