Accuzol reagent kit

Total RNAs were extracted from the EDTA-treated blood samples according to the manufacturer’s instructions for AccuZol™ reagent kit (Bioneer, Korea). Briefly, 250 μL blood sample was added to 750 μL AccuZol™ in a 1.5-mL Eppendorf tube and suspended several times by vortexing. Chloroform (200 μL) was added to each sample, and the mixtures were vortexed vigorously for 15 s, incubated on ice for 5 min, and centrifuged at 16,000 × g for 15 min at 4°C. The resulted supernatant was aspirated and placed into 1.5 mL sterile tube, and an equal volume of isopropyl alcohol was added. The mixture was inverted, incubated at −20°C for 10 min, centrifuged at 16,000 × g for 10 min, added with 1 mL of 80% ethanol, and mixed again by vortexing. After centrifuging again at 16,000 × g for 5 min at 4°C, the supernatant was removed, and the pellet of RNAs retrieved was dissolved in RNase-free water, incubated at 60°C for 10 min, and deep-frozen.

The mRNA of tissue was extracted using the AccuZol® reagent kit (Bioneer) according to the manufacturer’s instructions. Briefly, 200 mg of tissue was weighed and placed in a 1.5 mL Eppendorf Tube, and 1 mL ofAccuZol reagent (Bioneer) was added. Subsequently, 200 μL of chloroform (Chem-Lab, Belgium) was added to each tube and shaken robustly for 15 seconds. Then, the mixture was incubated on ice for 5 min and centrifuged (Thermo Fisher Scientific, Germany) at 14,000× g at 4°C for 15 min. The pellets were neglected, and the supernatant was removed in the Eppendorf tube (Abdos LabTech, India) and added with 500 μL of isopropanol. Then, the mixture was mixed by inverting the tube 4-5 times and incubated at 4°C for 10 min. The samples were centrifuged at 14,000× g at 4°C for 10 min. The supernatant was poured off, added 1 mL of 80% ethanol, and mixed again using a vortex. Subsequently, the samples were recentrifuged at 14,000× g at 4°C for 10 min. The supernatant was neglected, and the RNA pellet yield was exposed to air for drying. Finally, the RNA pellets were loaded with 50 μL of diethylpyrocarbonate water (Bioneer) to each sample to dissolve them; then, these were stored in a freezer at −20°C. A NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) was used to evaluate the RNA concentration.