Materials and Methods🔗

Blood sample collection and RNA storing: Five milliliters of blood was collected from volunteers in EDTA vacutainers, mixed with 25ml of Erythrocyte Lysis Buffer, Qiagen, and incubated for 10 minutes on ice then mixed by vortexing briefly 2 times during incubation and kept for 10-20 minutes and centrifuged at 4000 rpm for

10 minutes at 4°C and the supernatant was completely removed and discarded. After 10-20 minutes visible pellets were re-suspended in 10ml of solution.

RNA Precipitation Procedure:

• 1. Add 1/10 volume 3M NaOH, pH 5.2 and 2.5 volumes ethanol.
• 2. Mix and incubate at -20°C for at least 1 hour.
• 3. Centrifuge at ≥ 12,000 x g in a microcentrifuge for 20 minutes at 4°C.
• 4. Wash pellet twice with 80% ethanol.
• 5. Air dry pellet. Check for dryness before proceeding.
• 6. Re-suspend pellet in DEPC-treated water.