RNA Extraction from Homogenized Tissue

Homogenized tissue samples (100 mg) were transferred into 1 mL of TRIzol in a 2 mL microcentrifuge tube. A total of 10 µL of fresh BME solution was added, mixed well by vigorous shaking/vortexing, and incubated for 5 min at room temperature. The tube was centrifuged at 12,000 rpm for 10 min at 4 °C. The aqueous phase was transferred into a 1.5 mL microcentrifuge tube and 200 µL of chloroform was added. The sample was mixed well, incubated for 5 min at room temperature, and centrifuged for 10 min at 12,000 rpm for phase separation. The aqueous phase was transferred into a 1.5 mL microcentrifuge tube and 0.7 volume of chilled isopropanol was added. The solution was mixed well by inversion and was incubated for 1 h at −20 °C. The supernatant was decanted after centrifugation at 12,000 rpm for 10 min at 4 °C. The pellet was washed with 70% ethanol. The supernatant was gently poured off or removed using a pipette. This step was repeated. The pellet was air dried and eluted in 50 µL of RNase-free water.