Nakao Reiko, Okauchi Hiroki, Hashimoto Chiaki, Wada Naoyuki, Oishi Katsutaka
Biological Clock Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Biological Clock Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan; Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, Japan.
Mol Genet Metab. 2017 Jun;121(2):190-197. doi: 10.1016/j.ymgme.2017.04.001. Epub 2017 Apr 5.
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.
实时逆转录-聚合酶链反应（RT-PCR）分析是一种用于测量mRNA表达水平的常用方法，也是基础研究的关键工具。鉴定稳定且不受实验条件影响的合适内参基因是RT-PCR准确定量的关键步骤。另一方面，许多转录本的水平在各种外周组织中呈现昼夜节律振荡，并且除了分子生物钟外，还被认为受进食节律调节。在此，我们研究了进食时间对在睡眠期或活动期进食的小鼠代谢组织中13种常见管家基因的时间表达谱的影响。这些基因中的大多数在肝脏和白色脂肪组织中的表达随进食节律而波动，但在骨骼肌中并非如此。双向方差分析（ANOVA）确定18S核糖体RNA（Rn18s）是肝脏和白色脂肪组织中唯一不受进食时间影响且全天稳定表达的基因，尽管RefFinder软件显示肽基脯氨酰异构酶A（Ppia）是最稳定表达的管家基因。ANOVA和RefFinder软件均确定Actb是骨骼肌的首选内参基因。此外，NormFinder提出肝脏中最佳的内参基因对是β-2微球蛋白（B2m）-Ppia，白色脂肪组织中是Ppia-TATA盒结合蛋白（Tbp），骨骼肌中是酪氨酸3-单加氧酶/色氨酸5-单加氧酶激活蛋白ζ多肽（Ywhaz）-甘油醛-3-磷酸脱氢酶（Gapdh），并且它们的稳定性值优于单个稳定基因。在小鼠昼夜节律研究中，用于标准化目标基因的合适内参基因对是肝脏中的B2m-Ppia，白色脂肪组织中的Ppia-Tbp，以及骨骼肌中的Ywhaz-Gapdh。
