Selection of suitable reference genes for core clock gene expression analysis by real-time qPCR in rat ovary granulosa cells
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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.
KeywordsClock genes Reference genes GeNorm NormFinder BestKeeper
We are grateful to Dr. Masa-aki Hattori (Kyushu University, Fukuoka, Japan) for the supply of the rats and Dr. Shimin Liu (UWA, Perth, Australia) for suggestions for modifying the paper. This study was supported by the National Key R&D Program of China (Project No. 2017YFD0502200).
Chuanjiang Cai designed the experiments and prepared the manuscript. Pengpeng Cai performed the experiments and the statistical analysis. Guiyan Chu coordinated all activities of this project and revised the manuscript. The corresponding author ensured that all co-authors had checked and confirmed their contribution statement prior to manuscript submission.
Compliance with ethical standards
Conflict of interest
Chuanjiang Cai, Pengpeng Cai, Guiyan Chu declare that they have no conflict of interest.
All of the experiments were performed under the control of the Guidelines for Animal Experiments in the Faculty of Medicine, Kyushu University, and in compliance with Law No. 105 and Notification No. 6 of the Government of Japan.
Additional informed consent was obtained from all individual participants for whom identifying information was included in this article.
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