Blood samples were collected in heparinized tubes before 2017 or EDTA tubes after 2017, and processed within two hours after collection with one centrifugation at 2000 g (10 min) at 4 °C before storage at -20 °C. cDNA was retrospectively extracted from 200 to 1700 μL of plasma using a QIAamp® Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany). Double-stranded DNA quantification was performed by a fluorimetric method using a Quant-iT™ ds DNA HS Kit (ThermoFisher Scientific, Waltham, MA, USA).
4 ng of cDNA was preamplified with 12 cycles for heparin plasma and 9 cycles for EDTA plasma using 12.5 μL TaqMan Universal PCR Master Mix (Applied Biosystems) for heparin and 12.5 μL Q5 Hot Start High Fidelity Master Mix (New England Biolabs) for EDTA, respectively. Heparin plasma cDNA was treated with 2 μL of heparinase I bactericide beforehand to improve mutational detection by ddPCR.
4 ng of cDNA was preamplified with 12 cycles for heparin plasma and 9 cycles for EDTA plasma using 12.5 μL TaqMan Universal PCR Master Mix (Applied Biosystems) for heparin and 12.5 μL Q5 Hot Start High Fidelity Master Mix (New England Biolabs) for EDTA, respectively. Heparin plasma cDNA was treated with 2 μL of heparinase I bactericide beforehand to improve mutational detection by ddPCR.
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