Quantitative Real-Time PCR Protocol

Total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Reverse transcription was carried out using the PrimeScript RT Master Mix (Takara). qPCRs were performed on a Rotor-Gene Q machine (QIAGEN) with SYBR Green Realtime PCR Master Mix (TOYOBO). The primers were designed according to previously reported methods28 (link)39 (link) and obtained as amplimer sets from Sangon Biotech (Shanghai, China). The sequences of the primers were as follows: GAPDH forward: 5′-TGACCACAGTCCATGCCATC-3′, GAPDH reverse: 5′-GACGGACACATTGGGGGTAG-3′; Nr4a1 forward: 5′-CGTTATCCGAAAGTGGGCAG-3′, Nr4a1 reverse: 5′-CGGGTTTAGATCGGTATGCCAGC-3′; and SNRPN forward: 5′-GCAAAACAGCCAGAACGTGAA-3′, SNRPN reverse: 5′-GCACACGAGCAATGCCAGTAT-3′. Data analysis was conducted by using the comparative Ct method in software by QIAGEN, and the results were normalized to GAPDH.