E z n a tissue rna kit

Samples at 0.5 cm³ in volume were obtained from the head kidney, placed in tubes with RNAlater® and stored at −80°C until further analysis. All tissue samples were placed in microtubes with 1 mL TRIzol and ceramic beads and homogenized in a BeadBug® Microtube Homogenizer (Benchmark Scientific, Edison, NJ, USA) at room temperature. Then, 200 μL of chloroform:isoamyl alcohol was added, vigorously mixed and allowed to stand for 2 min before centrifuging at 4°C for 15 min at 12,000 G. The supernatant was transferred to a new tube and mixed with 400 μL of 70% ethanol. This mixture was passed through the columns with the E.Z.N.A.® Tissue RNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA) according to the manufacturer's instructions for RNA extraction. Total RNA was quantified using the fluorimetry method in a Qubit^TM 3.0 Fluorometer (Invitrogen^TM, Thermo Fisher Scientific, Wilmington, DE, USA), and the quality of the total RNA was determined by visualizing the RNA bands separated by electrophoresis in 1% agarose gels using a FlashGel^TM System (Lonza Group, Allendale, NJ, USA).

The cholesterol assay kit was purchased from Randox Laboratories Co., Ltd. (Antrim, UK). The E.Z.N.A™ tissue RNA kit was from Omega Bio-Tek (Norcross, GA, USA). The high-capacity RNA-to-cDNA kit and the power SYBR^® Green RT-PCR reagent kit were obtained from Applied Biosystems (Richmond, CA, USA). The TG-GPO liquid reagent set was obtained from Teco Diagnostics (Anaheim, CA, USA).