Pyromark q24 advanced

PSQ was performed using the PyroMark Q24 Vacuum Workstation and PyroMark Q24 Advanced instrument with PyroMark Q24 Advanced CpG Reagents and PyroMark Q24 Advanced Accessories (all Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Primer sequences, sequences to analyze, and dispensation orders are listed in Table 1. DNA immobilization was optimized in a range from 5–39 µL biotinylated PCR-HRM product (pool of technical replicates) with 1 µL Streptavidin Sepharose High Perfomance beads (GE Healthcare, Germany) and 40 μL PyroMark Binding Buffer per 80 µL immobilization reaction. 22.5 µL biotinylated PCR-HRM product, 1.5 µL Streptavidin Sepharose High Performance beads, and 60 μL PyroMark Binding Buffer per 120 µL immobilization reaction was also tested. DNA immobilization was performed under agitation for 10 min at 1400 rpm. The captured PCR product was denatured, washed, and finally the biotinylated strand was transferred into a PyroMark Q24 Plate containing 20 µL of 0.375 μM sequencing primer in PyroMark Annealing Buffer. The plate was heated at 80 °C for 5 min and then transferred into the instrument holding the PyroMark Q24 Cartridge loaded according to pre-run information provided by the PyroMark Q24 Advanced software 3.0.0 (Qiagen, Hilden, Germany).

Each sample was analyzed in duplicate. PyroMark Q24 was used to determine the methylation rate at each CpG site, and then methylation rates were accurately analyzed with PyroMark Q24 Advanced software, version 3.0.0 (Qiagen).

For pyrosequencing, 500 ng of gDNA was subjected to bisulfite conversion using the EpiTect bisulfite kit (Qiagen). PCR reactions were performed on 12.5 ng of bisulfite-treated DNA in a final volume of 25 µL using the Pyromark PCR kit (Qiagen) with one of the primers being biotinylated for later capture. The primers were designed using the PyroMark assay design software 2.0 (Qiagen) (Supplemental Table S10). The initial denaturation/activation step was performed for 15 min at 95°C, followed by 50 cycles of 30 sec at 94°C, 30 sec at 54°C, 45 sec at 72°C, and a final extension step for 10 min at 72°C. The quality and the size of the PCR products were evaluated by running 5 µL of each PCR product on 1.5% (w/v) agarose gel in a 0.5× TBE buffer. The biotinylated PCR products were immobilized on streptavidin-coated Sepharose beads (GE Healthcare). DNA strands were separated using the PyroMark Q24 vacuum workstation; the biotinylated single strands were annealed with 0.375 µM sequencing primer (Supplemental Table S10) and used as a template for pyrosequencing. Pyrosequencing was performed using PyroMark Q24 advanced (Qiagen) according to the manufacturer's instructions, and data about methylation at each CpG were extracted and analyzed using the PyroMark Q24 advanced 3.0.0 software (Qiagen).