Quantus photometer

After determination of the DNA content using Quantus photometer and QuantiFluor dsDNA-System Kit (Promega, Mannheim, Germany), 80 ng of the purified DNA fragments from unmethylated genomic control DNA (EpiTect PCR Control DNA kit, Qiagen) after pre-amplification were bisulfite modified using EpiTect Fast DNA Bisulfite Kit (Qiagen GmbH) according to manufacturer's instructions. The resulting bisulfite modified fragments were eluted in 40 μl EB buffer and quantified with 9.72 × 10⁸ copies/μl (3.0 × 10⁸ copies/ng) of unmethylated PLA2R1 DNA fragments using ddPCR. Correspondingly, DNA fragments isolated from U937 cells and bisulfite modified methylated PLA2R1 were quantified with 6,500 copies/μl. After determination of the cfDNA content isolated from pooled serum samples ranging between 152 ng and 2,200 ng (Table 2) all the isolated cfDNA were bisulfite modified and the bisulfite-converted samples were eluted in 45 μl EB buffer and stored at – 80°C until analysis.

DNA concentration was determined using a Quantus photometer and QuantiFluor dsDNA-System Kit (Promega, Mannheim, Germany). Bisulphite modification was conducted using an EpiTect Fast DNA Bisulfite Kit (Qiagen GmbH, Düsseldorf, Germany) according to the manufacturer’s instructions with an elution volume of 40 µL EB buffer. Samples were concentrated and ethanol residues were reduced by incubating the bisulphite-treated DNA for 20 min at 70 °C, reaching a final sample volume of 12 µL. Samples were stored at −80 °C until further analysis.

DNA concentration was determined using Quantus photometer and QuantiFluor dsDNA-System Kit (Promega, Mannheim, Germany) and bisulfite modification was conducted with EpiTect Fast DNA Bisulfite Kit (Qiagen GmbH, Düsseldorf, Germany) according to manufacturer’s instructions with an elution volume of 40 µL EB buffer. Samples were stored at −80 °C until analysis.