Quantitative Real-Time PCR Analysis

Total RNA was extracted using ISOGEN kits (Nippon Gene, Tokyo, Japan). cDNA synthesis and qRT-PCR were performed as previously described [45 (link)]. Briefly, 300 ng of total RNA was reverse transcribed using random hexamers (Takara Bio) and Moloney murine leukemia virus reverse-transcriptase (Thermo Fisher Scientific). Of each cDNA, 10 ng was added to the SYBR Green Realtime PCR Master Mix (Toyobo, Osaka, Japan) or LightCycler 480 SYBR Green I Master (Roche, Basel, Switzerland) with 1 µM of each primer (see Table S1). Real-time fluorescence monitoring was performed using LightCycler 2.0 or LightCycler 480 II (Roche). Values were normalized to 18S rRNA, expressed relative to controls (treated empty vector (pSG5), small interfering RNA (siRNA) targeting green fluorescent protein (siGFP), or vehicle). The BMAL1 gene, a known RORα target, was used as a positive control.