Sybr 2

Total RNA was isolated using the RNA Fast 200 purification kit from Fastagen Biotech (Shanghai, China) according to the manufacturer’s instructions. Reverse transcription of RNA was performed with ExScriptTM RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). The cDNA was then subjected to RT-PCR. All primer pairs shown in Table II were designed by Oligo 6.0 primer analysis software (Oswel Research Products, Beijing, China). qPCR was performed in duplicate reactions of 12.5 μl volume SYBR II (Takara Biotechnology Co., Ltd.), 1 μl each of specific forward and reverse primers and 9.5 μl RNase Free H₂O up to 24 μl. Samples were heated for 85°C followed by 40 cycles of amplification for 15 sec at 95°C and 1 min at 60°C. The mRNA level of β-actin was measured as an internal reference.

Total RNA was extracted from all tested tissues with the EZ-10 DNAaway RNA Mini-prep Kit [Sangon Biotech (Shanghai), Co., Ltd], and then cDNA was synthesized from 1 µg RNA using the PrimeScript™ RT reagent kit with gDNA Eraser according to the manufacturer’s instructions (Perfect Real Time; TaKaRa Biotechnology, Dalian, China). The gene-specific primers for qRT-PCR of the candidate genes and reference gene are listed in Additional file 1: Table S7. The PCR consisted of 10 μL SYBR II (TakaRa), 2.0 μL cDNA, 1.6 μL primer, 0.4 μL ROX Reference Dye II and distilled water to a final volume of 20 μL. The PCR program was as follows: 95 °C for 30 s and 35 cycles of 95 °C for 5 s, followed by 56–60 °C (depending on the primers used) for 30 s. For each reaction, three biological replicates were performed, and relative expression levels were obtained using the 2^−∆∆Ct method, with BnActin7 as internal controls.