Ez 10 dnaaway rna mini prep kit

Total RNA was extracted using the EZ-10 DNAaway RNA Mini-prep Kit (Sangon Biotech, Shanghai, China). NanoDrop 2000 (Thermo Fisher Scientific, Worcester, MA, USA) and electrophoresis were used to measure concentrations and RNA integrity. Complementary DNA was obtained using the iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) and diluted 15 times with distilled deionized water for qRT-PCR. The composition of qRT-PCR contained 2 µL of 15-fold diluted cDNA solution, 10 µL of SYBR® Green Supermix (Bio-Rad), 0.4 µL of 10 mM forward and reverse primers and 7.2 µL of distilled deionized water. Primers were designed on Primer Premier Software (version 5.0) (Table S8) [56 (link)] and qRT-PCR was performed on a CFX96 Real-time System (Bio-Rad) with the following conditions: 98 °C for 30 s, then 40 cycles of 98 °C for 15 s, 55 °C for 30 s, and an increase from 65–95 °C at increments of 0.5°C every 0.05 s. Three biological replicates and three technical replications were used for qRT-PCR. According to the 2^−ΔΔCt method using Actin7 and UBC21 as internal controls, the gene expression levels were determined and displayed by OriginPro 8 (OriginLab Corporation, Northampton, MA, USA).

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.