Serum was separated by centrifugation immediately after clotting and stored at −20 °C until further processing. Frozen serum was thawed at 4 °C, and cell debris was removed by centrifugation at 4000 × g for 20 minutes. Total nucleic acids were extracted from 200 μL of the supernatant using High Pure Nucleic Acids Extraction Kit (Roche) according to manufacturer’s instructions.