Pyromark q24 md system

Bisulfite pyrosequencing was performed as described previously [26 (link)]. Briefly, bisulfite-treated DNA was amplified using a PyroMark PCR kit (Qiagen, Hilden, Germany). PCR and cycling conditions were according to the kit manual. All pyrosequencing primers (PCR primers and sequencing primers) were based on the selected candidate 450 K array CpG probe using PyroMark Assay Design software (Qiagen). The amplification protocol was performed according to Collela et al. [27 (link)] using a universal primer approach. The biotinylated PCR products were captured using 1.0 μl streptavidin-coated sepharose high-performance beads (GE Healthcare, Little Chalfont, UK). The immobilized products were washed with 70 % alcohol, denatured with PyroMark denaturation solution (Qiagen), and then washed with PyroMark wash buffer (Qiagen). The purified PCR product was then added to 25 μl PyroMark annealing buffer (Qiagen) containing 0.3 μM sequencing primers for specific genes (all primers and their sequences are available on request). Finally, pyrosequencing was performed using the Pyromark Q24 MD system (Qiagen) according to the manufacturer’s instructions using the PyroGold Q24™ Reagent kit (Qiagen). Data were analyzed and quantified with the PyroMark Q24 software version 2.0.6 (Qiagen).

To confirm that methylation-specific PCR specifically amplified the methylated allele, the methylation status of the PCR product was quantitatively sequenced using pyrosequencing as previously described 23 (link). Briefly, the biotinylated PCR product was bound to streptavidin Sepharose beads HP (GE Healthcare Life Sciences), washed and denatured using a 0.2 mol/L NaOH solution. After addition of 0.3 μmol/L sequencing primer to the purified, single-stranded PCR product, pyrosequencing was carried on PyroMark Q24 MD System (Qiagen) with Pyro Q-CpG software (Qiagen) according to the manufacturer's instructions. Primer sequences and conditions, and the number of analyzed CpG sites are shown in Table 1. Methylation control samples (0% and 100%) were analyzed in every assay to check that no PCR product was obtained in the 0% control sample and that the fully methylated allele was amplified in the 100% control sample.