Pyromark q24 advanced accessories

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).

PSQ was performed using the PyroMark Q24 Vacuum Workstation and PyroMark Q24 Advanced instrument with PyroMark Q24 Advanced Accessories, Pyro-Mark Q24 Advanced CpG Reagents (all Qiagen), and Sepharose High Performance beads (GE Healthcare; Thermo Fisher Scientific) according to the manufacturer’s instructions. Dispensation orders were published previously [9 (link)]. PSQ data were evaluated and exported with the PyroMark Q24 Advanced software 3.0.0 (Qiagen). DNA methylation levels obtained by PSQ ≤ 5% (lower limit of quantification, LLOQ) and ≥95% (upper limit of quantification, ULOQ) were substituted with default values, namely, 2.5% and 97.5%, respectively, as proposed previously [36 ].
For better comparability of HRM and PSQ data, PSQ assays were calibrated. The main aim was to eliminate differences due to PCR bias. Calibration was conducted for each CpG separately using R based on signals for unmethylated and methylated DNA standards and their 25%, 50%, and 75% mixtures.