Gel electrophoresis and densitometry analysis

MSP analysis was carried out by running 5 μL PCR amplicon products on 2 % agarose gel. The gels were scanned on Fusion SL (Vilber Lourmat, Marne la Vallee, France) and visualized using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). In order to quantify the ratio between methylated and unmethylated bands, samples with saturated bands were re-run with a lower concentration ratio of DNA template for both methylated and unmethylated PCRs.

The methylated and unmethylated band intensities were quantified using ImageJ software and the ratio between methylated to unmethylated was calculated for each sample using Microsoft Excel (Microsoft Corporation, Redmond, Washington, USA). A standard rectangular-frame was estimated according to the size of the smallest band in a given gel. Consequently, the same rectangular-frame was used to measure the intensity of each band within the same gel to provide consistency. The measurement was set at integrated density to calculate the intensity value of the band based on the amount of amplicon present. All quantifications were carried out by two independent researchers to minimise observational errors.