Title : Comprehensive evaluation of blood-brain barrier-forming micro-vasculatures: Reference and marker genes with cellular composition
Primary brain microvessels (BrMV) maintain the cellular characters and molecular signatures as displayed in vivo, thus serve as a vital tool for biomedical research of the blood-brain barrier (BBB) and the development/optimization of brain drug delivery. The variations of relative purities or cellular composition among different BrMV samples may have significant consequences in data interpretation and research outcome, especially for experiments with high-throughput genomics and proteomics technologies. In this study, we aimed to identify suitable reference gene (RG) for accurate normalization of real-time RT-qPCR analysis, and determine the proper marker genes (MG) for relative purity assessment in BrMV samples. Out of five housekeeping genes, ß-actin was selected as the most suitable RG that was validated by quantifying mRNA levels of -L-iduronidase in BrMV isolated from mice with one or two expressing alleles. Four marker genes highly/selectively expressed in BBB-forming capillary endothelial cells were evaluated by RT-qPCR for purity assessment, resulting in Cldn5 and Pecam1 as most suitable MGs that were further confirmed by immunofluorescent analysis of cellular components. Plvap proved to be an indicator gene for the presence of fenestrated vessels in BrMV samples. These methods open the door to more accurate investigations of the BBB for determining changes in physiological and relevant clinical conditions, and for developing RNA-Seq-based molecular atlas of the BBB in animals and humans for new targets, signaling pathways within the neurovascular unit, and new therapies. This study may contribute to the building blocks toward overarching research needs to surmount challenges around the interface between the blood and the brain.