Methylated dna quantification kit

Genomic DNA was extracted from mice kidneys as previously described (Chomczynski 1993) ; samples were incubated in 150 µl of 100% ethanol (RT, 3 min) prior to centrifugation (2000×g, 4 °C, 5 min), and the resultant supernatant was discarded. The pellets were incubated in 500 µl of 0.1 M sodium citrate in 10% ethanol (pH 8.5) (RT, 30 min) and centrifuged (2000×g, 4 °C, 5 min); this was repeated twice before being re-suspended in 1000 µl of 75% ethanol and incubated (RT, 15 min). The pellets were centrifuged (2000×g, 4 °C, 5 min), air dried (RT, 5 min), re-suspended in 300 µl of 8 mM sodium hydroxide buffer, and centrifuged again (12,000×g, 4 °C, 10 min). The concentration of the DNA was measured using the Nanodrop spectrophotometer (ND-2000; Thermo-Fisher Scientific) and standardized to 500 ng/µl. DNA purity was determined through the A260/A280 absorbance ratio.
Global DNA methylation was quantified using the Methylated DNA Quantification Kit (Abcam, ab117128), following the manufacturer's protocol. The absorbance was measured at 450 nm on a spectrophotometer (SPECTROstar nano spectrophotometer, BMG Labtech). The percentage of 5-methylcytosine (5-mC) in total DNA was determined using the supplied formula (Supplementary Material) and expressed as a relative foldchange in comparison to the control.

Total DNA was prepared using the ZR-Duet™ DNA/ RNA MiniPrep (Zymo Research) according to the manufacturer's protocol. For the measurement of global DNA methylation status, Methylated DNA Quantification Kit was used (Abcam). To quantify the absolute amount of methylated DNA a standard curve was generated (0.5, 1.0, 2.0, 5.0 and 10.0 ng/µl for 5-mC), and the OD (optical density) values versus the amount of positive control at each concentration point were plotted. The slope (OD/ng) of the standard curve was determined using linear regression. The amount (ng) of methylated DNA (5-mC) was calculated by subtracting the negative control OD from sample OD and divided by the slope (OD/ng) and multiplied by 2, which is a factor to normalize 5-mC in the positive control to 100%, as the positive control contains only 50% of 5-mC, according to the manufacturer's protocol. The percentage of methylated DNA was calculated by dividing the amount of 5-mC (ng) by the amount of input sample DNA (ng) and multiplying by 100%. Absorbance at 450 nm was measured using with ELISA plate reader (Dynex Technologies Triad Multi-Mode Microplate Reader). Samples were prepared in triplicate.