H-FIRE BBBD Mechanisms Normal Rat Brain Data
Raw SuperArray and qRT-PCR data for study investigating the mechanisms of H-FIRE-mediated BBBD in normal rat brains.
Steps to reproduce
Total RNA from brains following H-FIRE treatment was extracted using the Qiagen AllPrep DNA/RNA/Protein Mini Kit and Qiagen RNeasy Mini Kit following the manufacturer’s protocols. Briefly, brain tissue samples weighing no more than 30g were collected from within the treatment region of transverse brain sections as identified by the presence of Evan’s blue dye within the brain parenchyma. Brain tissue samples were placed in microcentrifuge tubes containing 600uL of Buffer RLT Plus (10uL -mercaptoethanol per 1 mL Buffer RLT) and manually homogenized using a RNase-free disposable pellet pestle (Fisherbrand). Total RNA was eluted using 30uL of RNase-free water and quantified using OD260 on a NanoDrop 2000 microvolume spectrophotometer (Thermo Scientific, Baltimore, MD, USA). Total RNA (540 ng) was pooled from 2-4 individual rats representing each post-treatment time point for cDNA synthesis via RT2 Easy First Strand Kit (Qiagen). Analyses were performed in triplicates using the RT2 Profiler PCR Array Rat Tight Junctions Platform (Qiagen) according to the manufacturer’s protocol using an ABI Fast Block 7500 thermocycler (Applied Biosystems, Memphis, TN, USA). Gene expression data was analyzed using the manufacturers online software, Gene Globe (Qiagen) and Ingenuity Pathways Analysis (IPA). The mean Ct value from triplicate reactions was used for fold-change analysis, which was calculated relative to sham controls. IPA data were ranked and evaluated based on z-score. Genes with the greatest differential expression based on z-scores generated by GeneGlobe and IPA analysis results were further evaluated with TaqMan qRT-PCR to validate our initial findings. These included Cldn5, Cldn6, Cldn11, Cldn12 and Gnai2. Total RNA (800 ng) isolated from individual rats (no pooling) representing each post-treatment time point was used for cDNA synthesis via the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher) according to the manufacturer’s instructions. All primers were supplied by ThermoFisher. A master mix was prepared for each individual primer and contained the following per planned reaction: 7uL nuclease-free water, 10uL Taqman Fast Universal PCR Master Mix (ThermoFisher), and 1uL of primer. Triplicate PCR reactions were prepared in a 96-well plate by combining 18uL of Master Mix and 2 uL of cDNA to each well. Quantitative real-time PCR was completed using an ABI Fast Block 7500 thermocycler. Mean Ct values from triplicate experiments were used to calculate fold change in gene expression relative to sham controls. Lactate dehydrogenase A (Ldha) was used as the housekeeping gene.