Qcpg software

Methylation levels were quantified using pyrosequencing (EpigenDx, Hopkiton, MA, USA), to examine the methylation status at four CpG sites in the GADD45a 5′-UTR located between –737 and −690 relative to the transcription start site. Bisulphite-treated DNA template was amplified by nested PCR. The outer PCR product of 116 bp was generated using primers, forward: 5′-TTGGGTTGTTAGGGATTTTTATATG-3′ and reverse: 5′-AAAATCTTTTCCACAAAAAACAAAA-3′. This was then purified using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) after agarose gel electrophoresis. The resulting DNA was amplified using forward primer: 5′-TGGGTTGTTAGGGATTTTTATATG-3′ and the biotinylated reverse primer: 5′-AAAATCTTTTCCACAAAAAACA-3′. The biotinylated primer was used to capture one single-stranded DNA template for pyrosequencing (Royo et al, 2007 (link)). The PCR products (10 μl) were sequenced using the forward sequencing primer: 5′-GATTTTTATATGTGGTTATT-3′ on the Pyrosequencing PSQ96 HS System (Biotage, Uppsala, Sweden). Loci were analysed as a T/C SNP using QCpG software (Biotage). Percent methylation was calculated as the relative peak height of T vs C at each CpG site. Each plate contained unmethylated, partially methylated and heavily methylated DNAs (SssI-treated DNA) for quality control.