Transcriptome Analysis of Spinal Cord at Cytokine Injection Site

Transcriptional analysis was performed at 14 DPI. Spinal cords were sampled with a width of 4.5 mm (centered on cytokine injection site) after transcardial perfusion with ice-cold PBS, then immediately frozen with liquid nitrogen and stored at −80°C until extraction. Total RNA extraction was performed using the Maxwell^® RSC simplyRNA Tissue Kit (Promega) according to the manufacturer’s protocol. Microarray analysis was performed using Mouse Genome 430 2.0 Array (Thermo Fisher Scientific) according to the manufacturer’s protocol. Single array analysis was performed using Microarray Suite version 5.0 (MAS5.0) and global scaling as the normalization method. Differential expression analysis and gene set enrichment analysis (GSEA) were performed using R-packages limma (28 (link)) and clusterProfiler (29 (link)) respectively. The gene set libraries for GSEA (Supplementary File 1) were prepared from Enrichr (30 (link)), CellMarker (31 (link)), and Munji et al. reported genes (32 (link)).

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Hirata T., Itokazu T., Sasaki A., Sugihara F, & Yamashita T. (2022). Humanized Anti-RGMa Antibody Treatment Promotes Repair of Blood-Spinal Cord Barrier Under Autoimmune Encephalomyelitis in Mice. Frontiers in Immunology, 13, 870126.

Publication 2022

Maxwell® RSC simplyRNA Tissue Kit

Cold Cytokine Frozen Gene libraries Genes Genome Microarray Mouse Nitrogen Perfusion Promega Spinal cords Tissue Transcriptional

Corresponding Organization : Osaka International University

Other organizations : Mitsubishi Group (Japan)

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Variable analysis

independent variables

Time after cytokine injection (14 DPI)

dependent variables

Transcriptional profiles (microarray analysis)

control variables

Sampling location (4.5 mm width centered on cytokine injection site)
Tissue collection (transcardial perfusion with ice-cold PBS, immediately frozen in liquid nitrogen and stored at -80°C)
RNA extraction (Maxwell® RSC simplyRNA Tissue Kit)
Microarray analysis (Mouse Genome 430 2.0 Array, MAS5.0 normalization)

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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