MethylFlash™ Methylated DNA Quantification Kit

Protocol tips

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	Do not allow pipette tip to touch the outer edges or inner sides of the wells to prevent solutions from sticking to the surface

Protocol tips
Do not allow pipette tip to touch the outer edges or inner sides of the wells to prevent solutions from sticking to the surface

Publication protocol

Genomic DNA from 25 mg of brain tissue was purified with the DNeasy blood & tissue kit (Qiagen, Inc). DNA concentration and purity was determined by comparing the ratio of optical density measurements at 260 and 280 nm. Global DNA methylation was determined using the Methyl Flash Methylated DNA Quantification Kit (Colorimetric) (Epigentek Group Inc., New York, NY, USA) [14]. The kit measures the methyl-cytosine content as a percentage of total cytosine content. Using a DNA concentration of 20 μg/mL, the purified DNA was added to ELISA plate. The methylated fraction of DNA is quantified using 5-methylcytosine specific antibodies. The amount of methylated DNA was proportional to the OD intensity in an ELISA plate reader at 450 nm. DNA methylation was calculated using the formula: [(sample OD–M3OD)/S]/[((M4OD– M3OD)x2)/P]x100; where OD is optical density; M3 is the negative control, an unmethylated polynucleotide containing 50% of cytosine; S is the amount of input sample DNA in ng; M4 is the positive control, a methylated polynucleotide containing 50% of 5-methylcystosine; P is the amount of input positive control in ng. The amount of methylated DNA was expressed as percentage of total DNA.

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Manufacturer protocol

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