Qiaamp minelute ccfdna mini kit

Genomic DNA was extracted from buffy coat using the QIAamp DNA Blood Mini Kit (51104, QIAGEN) according to the manufacturer’s instructions. cfDNA was extracted from 0.5 to 4 ml of plasma using the QIAamp MinElute ccfDNA Mini Kit (55204, QIAGEN) or the QIAamp Circulating Nucleic Acid Kit (55114, QIAGEN) according to manufacturer’s instructions. After isolation, DNA concentration was measured using the Qubit dsDNA HS kit (Q32851, Thermo Fisher Scientific), and size distribution was assessed using the Bioanalyzer instrument and DNA 1000 or HS DNA 1000 kit (5067-1504 or 5067-4626, Agilent).

Peripheral blood was collected from the veins of patients and centrifuged within 30 min (4℃, 3000 rpm, 10 min). The plasma was collected and stored at 4℃ for DNA extraction within 3 days, according to the instructions of the QIAamp MinElute ccfDNA Mini Kit (QIAGEN, Germany, 55204). The DNA was treated with bisulfite to convert cytosine to uracil according to the instructions of the EZ DNA Methylation-Gold Kit (Zymo Research, USA, D5001/D5003). Subsequently Qubit ssDNA HS Assay Kit (Invitrogen, USA, Q32854) as well as Qubit 3.0 fluorescence quantizer (Thermo Fisher, USA) were used for DNA quantitative analysis after bisulfite conversion. The bisulfite-converted DNA was stored at -20℃ until use.

DNA from fetal blood was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Germany) according to manufacturer instructions. DNA from fetal tissue was extracted using Puregene Cell and Tissue kit (Qiagen, Germany) according to manufacturer protocol. cffDNA from maternal plasma, serum and urine were extracted using the QiAamp MinElute ccfDNA mini Kit (Qiagen, Germany). Concentrations of extracted DNA was estimated using nanodrop (Nanodrop 8000, Thermo Scientific, DE, USA) and ensured the quantity >0.5 ng/µL and quality A260/A280 ratio of 1.8±0.2. Approximately 5% of the samples were quantified using Qubit 2.0 fluorometer (Qubit™ dsDNA HS Assay Kits; Life Technologies, CA, USA).

Total RNA‐free genomic DNA was extracted from the biopsy samples with RNase treatment using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. Tissue DNA was eluted with ultra‐clean water at a final elution volume of 50 µL. Plasma samples were analyzed to determine the OD at 414 nm (OD₄₁₄) using NanoDrop 2000 microvolume spectrophotometer (Thermo Fisher Scientific). A hemolysis cut‐off value was fixed at 0.13 (OD₄₁₄, 10 mm pathlength equivalent) with reference to the report by Pizzamiglio et al.¹⁷ Our preliminary data showed that hemolysis decreased the detectable methylation rates, because of DNA contamination from blood cells (Figure S1). Nonhemolytic plasma samples were chosen for DNA extraction. Circulating cell‐free DNA was extracted using the QIAamp MinElute ccfDNA Mini Kit (Qiagen) according to the manufacturer’s instructions. Two milliliters of plasma samples (1 mL/column) from each subject was used for DNA extraction. Each column of ccfDNA was eluted with 30 µL ultra‐clean water.

cfDNA was isolated from 2 ml of plasma using the QIAamp® MinElute® ccfDNA Mini Kit (Qiagen) according to the instructions of the manufacturer and recovered in 50 µl ultra-clean water. Concentration of cfDNA was then measured using the Qubit™ dsDNA HS Assay Kit with a Qubit™ 2.0 fluorometer (Invitrogen).