Quantifying miR-124a and BRD4 Levels

The levels of miR-124a and BRD4 in the whole blood and cells were measured by qRT-PCR. Total RNA was isolated from serum samples using a miRVana PARIS kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions. The cells treated with TRIzol reagent for extraction of total RNAs, and adherent cells were treated with trypsase before RNA extraction. After that, the total RNA in the extracts were transcribed as cDNAs by a PrimeScript® RT reagent Kit (Thermo Fisher, Massachusetts, USA). The primers of miR-124a and BRD4 synthesized by a PrimeScript® RT reagent Kit (Thermo Fisher, Massachusetts, USA) were used in the experiment. The reaction systems (10 μl) of qRT-PCR were prepared according to the operational instruction of a KAPA qRT-PCR kit (Sigma-Aldrich, Missouri, USA). U6 was used as the endogenous controls. The following conditions were used: denaturation at 95°C for 3 min, followed by amplification for 40 cycles at 95°C for 12 s and at 53°C for 40s, and 70°C for 30s. The relative levels of miRNA or mRNA were calculated with the 2^−(ΔΔCt) method [14 (link)]. The primer sequences of miR-124a, BRD4, and U6 are listed in Table 1.