Methylflash global dna methylation 5 mc elisa kit

DNA was isolated using the PureLink Genomic DNA Mini Kit (ThermoFisher Scientific). Six samples were collected to obtain 3 control and 3 FA treatment samples. The contents of two wells were used to obtain one sample—one well from one 6 well plate and one well from a separate 6 well plate. DNA was quantified using a nanodrop. Global DNA methylation was measured using a MethylFlash Global DNA Methylation (5-mC) ELISA Kit from Epigentek. The DNA samples were loaded in duplicate into the ELISA plate along with four methylated DNA standards. The DNA samples were incubated with an antibody for 5-methyl Cytosine (5-mC) for 1 h followed by washes and incubation with a secondary antibody with a conjugated reporter. The plates were then washed again and a substrate was added followed by a solution to inhibit the reporter-substrate reaction. Plates were then read at 450 nm using a plate reader. The percentage of 5-mC in each DNA sample was calculated according to the instructions provided in the Epigentek manual. A paired two-tailed t-test was utilized to determine significance between the 3 control replicates and the 3 FA treatment replicates.