Methylation Analysis of Imprinted Genes in Islets

DNA was extracted from pooled islets using the QIAmp DNA Mini Kit (Qiagen), and $1\mathrm{\mu }\mathrm{g}$ of DNA was bisulfite treated using the EpiTect Bisulfite Kit (Qiagen) following the manufacturer’s protocol. Pyrosequencing was conducted to measure the methylation status of the promoter region of the Igf2 DMR1 (chr7: 149,851,180–149,851,655; NCBI37/MM9) and H19/Igf2 ICR (chr7: 149,767,599–149,767,819; NCBI37/MM9), and exon A and exon C of the Esr1 gene as described previously by Susiarjo et al. (2013 (link)) and Kundakovic et al. (2013 (link)), respectively. Briefly, $50\text{ng}$ of purified bisulfite-treated DNA was amplified using a PyroMark PCR kit (Qiagen) and primers specific for the Igf2 DMR1, H19/Igf2 ICR, and exon A and exon C of the Esr1gene (see Table S2). Ten microliters of the biotinylated PCR product was used for each sequencing assay and sequenced using a PyroMark Q96MD (Qiagen) pyrosequencer and PyroMark Gold 96 reagents kit (Qiagen) and specific pyrosequencing primers (see Table S2). Percent methylation levels of CpG sites were quantified using Qiagen’s Pyro Q-CpG software.