Quantitative Expression Analysis of Circadian Genes

Isolation of total peripheral blood leukocytes, RNA extraction, cDNA synthesis, and qRT-PCR were performed as previously described (17 (link)). Briefly, the 2 μg RNA input for cDNA synthesis was determined by spectrophotometric OD260 measurement, and cDNA was generated with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), following the manufacture's protocols. Expression of the NR1D1 and RORC genes was analyzed using the TaqMan system, and all TaqMan Gene Expression Assays were purchased from Applied Biosystems. Expression of the human housekeeping gene, ACTB (actin beta), was used for normalizing NR1D1 and RORC expression in real-time qRT-PCR. Real-time qRT-PCR was performed in an ABI 7500 Fast Real-Time System (Applied Biosystems). The expression levels of the NR1D1, RORC, and BMAL1 were normalized to the internal control ACTB to obtain the relative threshold cycle (ΔCt).