Abstract
Primary brain microvessels (BrMV) maintain the cellular characters and molecular signatures as displayed in vivo, and 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, β-actin was selected as the most suitable RG that was validated by quantifying mRNA levels of alpha-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. This study may contribute to the building blocks toward overarching research needs on the blood-brain barrier.
Highlights
The blood-brain barrier (BBB) plays a key role in normal neuronal function, disease and aging as it serves the needs for homeostasis, entry of nutrients, and communication of the central nervous system (CNS)[1, 2]
The inter-assay variation determined by 9 PCR repeats using 3 RT products varied from 1.2% for Tbp and Hmbs to 1.8% for Pgk1, suggesting consistently low systematic errors for all RT-qPCR settings
When analyzing data with ΔΔCt methods for relative quantification of fold changes between sample sets (Fig 3C), Actb results in near-expected ratio (1.84), followed by Hmbs (1.70), Gapdh (1.67), and Tbp (1.31). These results show that expression profile of Gene of Interest (GOI) in isolated brain microvessels (BrMV) samples can be highly affected by the choice of reference gene (RG), validating Actb as the most suitable RG for sample normalization regardless of quantification analysis used
Summary
The blood-brain barrier (BBB) plays a key role in normal neuronal function, disease and aging as it serves the needs for homeostasis, entry of nutrients, and communication of the central nervous system (CNS)[1, 2]. BBB remodeling has been indicated in the pathogenesis of various CNS diseases such as neuronopathic lysosomal storage diseases or Alzheimer’s disease[3, 4]. The neuroprotective role of the BBB hinders rapid and wide systemic delivery of therapeutic drugs or diagnostic agents to the CNS[5]. The BBB comprises approximately 600 km of capillaries formed by highly specialized brain capillary endothelial cells (BrEC), which are characterized by less pinocytotic activity, a lack of fenestrations, and unique expression.
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