Ten milliliters of blood sample were drawn from each participant into an EDTA vacutainer tube. The samples were kept in a portable Styrofoam box with ice packs (0–4°C), processed within 6 hours, and stored at −80°C until RNA isolation was conducted.
Total RNA, including miRNA, was isolated and purified from plasma samples using Qiagen’s miRNeasy Serum/Plasma Kit (Qiagen, Valencia, CA), following the manufacturer’s instructions. To minimize cellular contamination, the plasma samples were centrifuged at 16,000×g at 4°C for 10 min and 200 μl supernatant was used for total RNA extraction. To control for variations in the starting material as well as the efficiency of the downstream total RNA extraction, 5 μl each of three synthetic spike-in RNA oligos (osa-miR414, cel-miR248, and ath-miR159a) were added at concentrations of 1 pg/μl for cel-miR248, 2 pg/μl for osa-miR414, and 4 pg/μl for ath-miR159a. Five microliters of total RNA from 25 μl total yield was used for miRNA expression analysis, which represented the RNA from 40 μl of plasma.
Total RNA, including miRNA, was isolated and purified from plasma samples using Qiagen’s miRNeasy Serum/Plasma Kit (Qiagen, Valencia, CA), following the manufacturer’s instructions. To minimize cellular contamination, the plasma samples were centrifuged at 16,000×g at 4°C for 10 min and 200 μl supernatant was used for total RNA extraction. To control for variations in the starting material as well as the efficiency of the downstream total RNA extraction, 5 μl each of three synthetic spike-in RNA oligos (osa-miR414, cel-miR248, and ath-miR159a) were added at concentrations of 1 pg/μl for cel-miR248, 2 pg/μl for osa-miR414, and 4 pg/μl for ath-miR159a. Five microliters of total RNA from 25 μl total yield was used for miRNA expression analysis, which represented the RNA from 40 μl of plasma.
