Selection of reference genes for studying blood-brain borders in the rat and the non-human primate using quantitative real-time PCR

Blood-brain borders, which are located at the endothelium of cerebral capillaries and the epithelium of choroid plexuses, play a central and complementary role in maintaining cerebral homeostasis, providing neuroprotection, and regulating neuro-immune interactions. The accuracy of gene expression measurements in these borders using quantitative real-time PCR depends on the reliability of the reference genes used for normalization. We investigated the stability of reference gene candidates in two species, the rat and the Macaca fascicularis monkey, under various experimental conditions typically encountered in brain border studies. Rps19 and ZRANB2 were identified as the most stable reference genes for the simultaneous comparison of brain capillaries, cerebral cortex, and choroid plexuses in rat and monkey, respectively. More stable genes were identified for comparative analyses of choroid plexuses from the lateral and fourth ventricles, such as Hmbs in the adult rat and RPL41 in the monkey. Uba52 and Rpl41 met the criteria for reliable reference genes in rat choroid plexus developmental studies under normal conditions and following fetal growth restriction. Hmbs and Hprt1 were suitable for studies on rat brain capillaries in the context of fetal growth restriction and chronic inflammation. Hmbs and ZRANB2 were selected for comparative analyses between cultured brain endothelial cells and isolated brain capillaries in rat and monkey, respectively. This study emphasizes that the selection of stable reference genes for blood-brain border studies depends heavily on the specific biological context and experimental design.