Computational protocol: Intrinsic Inflammation Is a Potential Anti-Epileptogenic Target in the Organotypic Hippocampal Slice Model

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Protocol publication

[…] At the end of the MEA recordings, cultured slices were collected and kept at − 80 °C until the assay was performed. We used RNeasy Plus Universal Mini kit (Qiagen, Antwerp, Belgium) to obtain total RNA from tissues. After homogenization of the slices by mixing with vortex in QIAzol Lysis Reagent, RNA was purified following the manufacturer’s instructions. RNA concentration was measured with a NanoDrop ND-1000 Spectrophotometer (ThermoFisher Scientific, Gent, Belgium). RNA quality was assessed using the Bio-Rad (Temse, Belgium) Experion Automated Electrophoresis System (RQI > 9). cDNA was then synthesized from 250 ng total RNA using Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, Gent, Belgium) in a total volume of 25 μl following the manufacturer’s protocol. Quantitative PCR reactions (qPCR) were performed using a CFX384 Real-Time System. Four times diluted cDNA (2 μl) was analyzed in duplicate for genes of interest (Suppl. Table ) expression using inventoried or made to order TaqMan® Gene Expression Assays (ThermoFisher Scientific, Gent, Belgium) and TaqMan® Universal PCR Master Mix (ThermoFisher Scientific, Gent, Belgium) in a final volume of 10 μl according to the manufacturer’s recommendations. Cq values were obtained from Bio-Rad CFX Manager 3.1 software using regression determination mode. Normalized relative expression levels were calculated using qbase+ software [] (Biogazelle NV, Gent, Belgium). Among 10 potential reference genes tested, PpiB and Hprt1 were identified with the geNormplus [] module in qbase+ as the most suitable reference genes and were used for normalization.For principal component analysis (PCA), we used normalized expression values from 20 genes (Suppl. Table ) at 3 time points (7, 14, and 21 DIV). PCA was performed separately for vehicle- or anti-TNFα antibody-treated slices and compared by graphical plotting of PC1/PC2 distribution. […]

Pipeline specifications

Software tools CFX Manager, qbase+
Application qPCR
Diseases Brain Injuries, Epilepsy, Epilepsy, Post-Traumatic
Chemicals Phenytoin