Evo m mlv rt premix ag11706

HBMEC were seeded in 60-mm dishes to 70–90% confluence and infected with C. sakazakii ATCC 29544 for 0, 1, 2, 3, and 4 h (MOI = 100). Total RNA was extracted from HBMEC using RNAeasy^TM Animal RNA Isolation Kit R0026 (Beyotime), and then reverse transcribed to cDNA using Evo M-MLV RT Premix AG11706 (Accurate Biotechnology, Changsha, China) with the suppliers’ instructions. Quantitative Real-Time PCR was performed with SYBR^® Green Premix Pro Taq HS qPCR Kit AG11701 (AG11701; Accurate Biotechnology) on a Bio-Rad iQ5 PCR system (Bio-Rad). The primer sequences for RT-PCR are shown in Table 1. ACTB was used as the control. The Ct values for RT-PCR samples were recorded. The relative mRNA levels were analyzed with the 2^−ΔΔCt method.

Total RNA was extracted from samples with the RNA-prep pure kit (Tiangen, Beijing, China) according to the instructions. Then, cDNA was synthesized from 500ng total RNA using Evo M-MLV RT Premix AG11706 (Accurate Biotechnology (Hunan) Co.,Ltd) in a 10 μL reaction volume. Using Oligo 7.0 software, primers for RT- qPCR were designed adhering to the instructions.