Total RNA Extraction and cDNA Synthesis

Total RNA was extracted from tissue samples using TRIzol total RNA extraction reagent (Takara, Dalian, China), according to the manufacturer's instructions. The integrity of total RNA was evaluated by 1% agarose gel electrophoresis in 6 × loading buffer (Takara, Dalian, China). The quantity and quality of total RNA was estimated using a Nanodrop 2000 Spectrophotometer with the OD₂₆₀ nm/OD₂₈₀ nm ratio expected to be between 1.8 and 2.0; meanwhile, the OD₂₆₀ nm/OD₂₃₀ nm ratio no less than 1.7 (Zhang et al., 2017 (link)). Samples were then stored at −80°C. Prime Script™ RT Reagent kit (Takara, Dalian, China) was used to synthesize first strand cDNA, according to the manufacturer's protocol. The resultant cDNA was stored at −20°C.