Revertaid master mix with dnase 1

A total of eight differentially expressed P450s from each treatment group were selected for qPCR validation. The relevant primers and internal reference gene (elongation factor-1 alpha, EF1α) information are shown in Table S1. RNA was extracted as described previously. The RevertAid™ Master Mix with DNase I (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to reverse transcribe these into cDNA. Quantitative PCR reactions were performed on a C1000™ Thermal Cycler PCR (Bio-Rad, Hercules, CA, USA) with a reaction volume of 20 μL per sample, including 1 μL cDNA template, 10 μL 2 × RealStar Green Fast Mixture (Genstar, Beijing, China), 1 μL forward and reverse primers, and 8 μL of ddH₂O. Finally, the expression levels of the target genes were calculated according to the method described by Pfaffl [32 (link)].

Total RNA from BV2 cells and Sp5 tissues were derived with TRIzol Reagent (Invitrogen, USA) and reversed to cDNAs using RevertAid Master Mix with DNase I (Thermo Fisher Scientific, USA). cDNA samples, gene specific primers (Table 1) and SYBR™ Green Master Mix (Thermo Fisher Scientific) were used to perform qPCR experiments according to the protocol, which were collected by ABI StepOnePlus PCR system (ABI-7500, Thermo Fisher Scientific). The relative mRNA expressions of genes were calculated by the 2^-△△CT method as described previously (18 (link)).