X dilution factor X 40) of the obtained RNA was determined by measuring the absorbance at 260 nm (A26o) and 280 nm (A2so).

Total RNA was isolated from cells usmg TRizol reagent following the manufacturer's protocol. Briefly, 2x106 cells were harvested by non-enzymatic cell dissociation buffer and washed once with PBS. The cell pellet was lysed with 1 mL ice-cold TRizol reagent. The lysate was centrifuged at 12,000 x g for 10 min at 4°C to pellet down cellular debris, polysaccharides, and high molecular weight DNA. The supernatant was gently decanted into a fresh microcentrifuge tube and 200 J.tL of chloroform /mL of TRizol was added and the tube was shaken vigorously for 15 s. The mixture was incubated at room temperature for 2-3 min before centrifugation at 12,000 x g for 15 min at 4°C. This resulted in the separation of the mixture into a lower organic phase and an upper aqueous phase. The aqueous phase containing the RNA was gently aspirated and transferred into a fresh microcentrifuge tube and 500 JlL of isopropanol /mL of TRizol reagent was added to precipitate the RNA. The mixture was centrifuged at 12,000 x g for 10 min at 4°C to isolate the RNA as a pellet. The supernatant was discarded and the pellet was washed once with 70% ethanol, centrifuged and the pellet was air-dried and re-dissolved in appropriate quantity of nuclease-free water. The purity (A26o/A2so > 1.8) and concentration (A260

Total RNA isolation

Molecular biology techniques