Determination of reference genes that are independent of feeding rhythms for circadian studies of mouse metabolic tissues.

Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis is a popular method for the measurement of mRNA expression level and is a critical tool for basic research. The identification of suitable reference genes that are stable and not affected by experimental conditions is a critical step in the accurate normalization of RT-PCR. On the other hand, the levels of numerous transcripts exhibit circadian oscillation in various peripheral tissues and it is thought to be regulated by feeding rhythms in addition to the molecular circadian clock. Here, we investigated the effects of feeding schedule on the temporal expression profiles of 13 common housekeeping genes in metabolic tissues of mice fed during either the sleep or the active phase. The expression of most of these genes fluctuated dependently on feeding rhythms in the liver and WAT, but not in skeletal muscle. Two-way analyses of variance (ANOVA) identified 18S ribosomal RNA (Rn18s) as the only gene that was stably expressed throughout the day independently of feeding schedules in the liver and WAT, although RefFinder software showed that peptidylprolyl isomerase A (Ppia) was the most stably expressed housekeeping gene. Both ANOVA and RefFinder software determined that Actb was the preferred reference gene for skeletal muscle. Furthermore, NormFinder proposed that the optimal pairs of reference genes were beta-2 microglobulin (B2m)-Ppia in the liver, Ppia-TATA box binding protein (Tbp) in WAT, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (Ywhaz)-glyceraldehyde-3-phosphate dehydrogenase (Gapdh) in skeletal muscle, and that their stability value was better than that of a single stable gene. The appropriate reference gene pairs for normalizing genes of interest in mouse circadian studies are B2m-Ppia in the liver, Ppia-Tbp in WAT, and Ywhaz-Gapdh in skeletal muscle.