Total RNA Isolation and cDNA Synthesis

Total RNA was extracted from tissue distribution and immune experiment samples using the RNAiso plus (TaKaRa, Japan) reagent following the vendor’s protocol. The concentration and quality of isolated RNA were measured using a NanoDrop 2000 (Thermo Fisher, USA) and electrophoresis on 1.5% agarose gels, respectively. Then, the RNA samples were processed with gDNA Eraser (TaKaRa, Japan) to eliminate genomic DNA contamination. First-strand cDNA was synthesized with 1 μg total RNA using the PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Japan) and PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Japan) according to instructions. The obtained cDNA was used for subsequent gene cloning and expression analysis. The cDNA mix was diluted to 1:10 before further experiments.