Qiazol

Total RNA was isolated from chicken muscle samples using QIAzol lysis reagent (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s instruction with a slight modification. Briefly, 200 mg of frozen chicken skeletal muscle was ground thoroughly into powder using a sterile mortar and pestle. The tissue powder was homogenized with 2.5 mL of QIAzol (Qiagen Inc., USA) for 40 s, and the homogenate was subsequently centrifuged at 12,000×g for 10 min, 4°C. The supernatant was mixed with 0.2 vol of chloroform and subsequently centrifuged at 12,000×g for 10 min at 4°C. The isolated RNA was precipitated by mixing 500 μL of the aqueous phase with an equal volume of isopropanol followed by centrifugation at 12,000×g for 8 min, 4°C. The RNA pellet was collected, washed with 75% ethanol and re-suspended in nuclease-free water. The isolated RNA was treated with DNase I (Thermo Fisher Scientific Inc., Waltham, MA, USA) following company’s protocol to remove any contaminated genomic DNA, and re-purified using QIAzol (Qiagen Inc., USA) according to the company’s instruction. Quantity of total RNA was measured using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., USA). Purity of the isolated RNA was determined according to relative absorbance at 260 nm to 280 nm (A₂₆₀/A₂₈₀), and at 260 nm to 230 nm (A₂₆₀/A₂₃₀).