Quest 5 hmc dna elisa kit

The 5hmC levels were measured in 375 DNA samples using Quest 5hmC^™ DNA ELISA Kits (Zymo Research, Boston, MA, USA). DNA (100 ng) from each patient or control was used for quantification. Biochemical assays were performed according to the manufacturer's recommendations and samples were read in a spectrophotometer at 450 nm. Absolute quantification was performed using the concentrations of positive control samples by the standard curve method. The amount of 5hmC was calculated from 100-ng samples of single-stranded DNA and is reported as a percentage (%).

Global DNA methylation and hydroxymethylation levels were measures using 5mC DNA and Quest 5hmC DNA ELISA Kits, respectively (Zymo Research, CA, USA). DNA was denatured at 98 °C for 5 min and then an input of 100 ng per treatment condition was used to run the assay per the manufacturer’s protocol. Briefly, the denatured DNA was incubated in the plate for 1 h at 37 °C followed by several washes and incubation with a mixture of the primary antibody (anti-5mC or anti-5hmC) and the secondary antibody for 1 h at 37 °C. Finally, HRP developer was added to each well, color development was allowed for 10–60 min at room temperature, and absorbance was measured at 405–450 nm using a multimode plate reader (M3 Molecular Devices, San Jose, CA, USA). The percentages of 5mC and 5hmC in each sample were calculated using the prediction equation generated by logarithmically plotting the absorbance values of the corresponding standard curve. A standard curve with known percentages of 5 mC was generated by mixing the supplied negative controls (0% methylation) and positive controls (100% methylation) in certain ratios recommended in the manufacturer’s protocol. For the 5hmC standard curve, five supplied control DNA sets with 5hmC percentages that ranged from 0% to 55% were used. All samples were run in triplicate and the data represent the average from three different experiments.