Laboratory of Molecular Mechanisms of Neuronal Interactions, I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia.
Laboratory of Neurobiology of the Brain Integrative Functions, I.P. Pavlov Department of Physiology, Institute of Experimental Medicine, St. Petersburg, Russia.
PLoS One. 2023 Feb 9;18(2):e0273224. doi: 10.1371/journal.pone.0273224. eCollection 2023.
Reverse transcription followed by quantitative (real-time) polymerase chain reaction (RT-qPCR) has become the gold standard in mRNA expression analysis. However, it requires an accurate choice of reference genes for adequate normalization. The aim of this study was to validate the reference genes for qPCR experiments in the brain of rats in the model of mild ketosis established through supplementation with medium-chain triglycerides (MCT) and intermittent fasting. This approach allows to reproduce certain neuroprotective effects of the classical ketogenic diet while avoiding its adverse effects. Ketogenic treatment targets multiple metabolic pathways, which may affect the reference gene expression. The standard chow of adult Wistar rats was supplemented with MCT (2 ml/kg orogastrically, during 6 h of fasting) or water (equivolume) for 1 month. The mRNA expression of 9 housekeeping genes (Actb, B2m, Gapdh, Hprt1, Pgk1, Ppia, Rpl13a, Sdha, Ywhaz) in the medial prefrontal cortex, dorsal and ventral hippocampus was measured by RT-qPCR. Using the RefFinder® online tool, we have found that the reference gene stability ranking strongly depended on the analyzed brain region. The most stably expressed reference genes were found to be Ppia, Actb, and Rpl13a in the medial prefrontal cortex; Rpl13a, Ywhaz, and Pgk1 in the dorsal hippocampus; Ywhaz, Sdha, and Ppia in the ventral hippocampus. The B2m was identified as an invalid reference gene in the ventral hippocampus, while Sdha, Actb, and Gapdh were unstable in the dorsal hippocampus. The stabilities of the examined reference genes were lower in the dorsal hippocampus compared to the ventral hippocampus and the medial prefrontal cortex. When normalized to the three most stably expressed reference genes, the Gapdh mRNA was upregulated, while the Sdha mRNA was downregulated in the medial prefrontal cortex of MCT-fed animals. Thus, the expression stability of reference genes strongly depends on the examined brain regions. The dorsal and ventral hippocampal areas differ in reference genes stability rankings, which should be taken into account in the RT-qPCR experimental design.
逆转录后定量（实时）聚合酶链反应（RT-qPCR）已成为 mRNA 表达分析的金标准。然而，它需要准确选择参考基因进行充分的归一化。本研究的目的是验证通过补充中链甘油三酯（MCT）和间歇性禁食在轻度酮症模型中大鼠大脑 qPCR 实验的参考基因。这种方法可以复制经典生酮饮食的某些神经保护作用，同时避免其不良反应。生酮治疗针对多种代谢途径，这可能会影响参考基因的表达。成年 Wistar 大鼠的标准饲料补充 MCT（经口 2 ml/kg，禁食 6 小时）或水（等体积）1 个月。通过 RT-qPCR 测量中前额皮质、背侧和腹侧海马中的 9 种管家基因（Actb、B2m、Gapdh、Hprt1、Pgk1、Ppia、Rpl13a、Sdha、Ywhaz）的 mRNA 表达。使用 RefFinder®在线工具，我们发现参考基因稳定性排名强烈依赖于分析的脑区。在中前额皮质中发现最稳定表达的参考基因是 Ppia、Actb 和 Rpl13a；在背侧海马中是 Rpl13a、Ywhaz 和 Pgk1；在腹侧海马中是 Ywhaz、Sdha 和 Ppia。B2m 被确定为腹侧海马中的无效参考基因，而 Sdha、Actb 和 Gapdh 在背侧海马中不稳定。与腹侧海马和中前额皮质相比，所检查的参考基因在背侧海马中的稳定性较低。当归一化为三个最稳定表达的参考基因时，MCT 喂养动物的中前额皮质中 Gapdh mRNA 上调，而 Sdha mRNA 下调。因此，参考基因的表达稳定性强烈依赖于被检查的脑区。背侧和腹侧海马区在参考基因稳定性排名上存在差异，在 RT-qPCR 实验设计中应考虑到这一点。
