Selection of suitable reference genes for core clock gene expression analysis by real-time qPCR in rat ovary granulosa cells.

Selection of a suitable endogenous reference gene is essential for investigating expression of clock genes Bmal1, Clock, Pers, Crys, Rev-erbα/β, and RORα/β/γ involved in the circadian system. In this study, we treated rat ovary granulosa cells with dexamethasone to synchronize circadian oscillation in vitro and determined expression levels of Bmal1 and Per2 and six candidate reference genes (Actb, Beta actin; B2m, Beta-2-microglobulin; Ppia, Cyclophilin A; Gapdh, Glyceraldehyde-3-phosphate dehydrogenase; Hprt, Hypoxanthine guanine phosphoribosyl transferase and Tbp, TATA-box-binding protein) using quantitative real-time PCR. We then employed three software programs, GeNorm, NormFinder, and BestKeeper, to analyze the expression data for the selection of the best reference gene. According to GeNorm, Tbp and B2m were assessed as the most stable reference genes; Tbp and Hprt were best by NormFinder and BestKeeper, respectively. Thus, we recommend Tbp as the most suitable reference gene for studying clock genes expression in rat ovary granulosa cells in vitro.