Page 149 - scheppingen
P. 149

MIR147B: A NOVEL KEY REGULATOR OF IL-1β-MEDIATED INFLAMMATION IN ASTROCYTES stimulation with IL-1β was initialized, and treatment was continued during stimulation.
RNA isolation
For RNA isolation, cell culture or fresh brain tissue was homogenized in Qiazol Lysis Reagent (Qiagen Benelux, Venlo, The Netherlands). Total RNA was isolated using the miRNeasy Mini kit (Qiagen Benelux, Venlo, The Netherlands) according to manufactur- er’s instructions. The concentration and purity of RNA were determined at 260/280 nm using a NanoDrop 2000 spectrophotometer (Ocean Optics, Dunedin, FL, USA).
RNA-Sequencing
RNA-Sequencing (RNA-Seq) was performed on control and IL-1β stimulated fetal astro- cytes (n = 5 donors). Two different RNA-Seq techniques were used; mRNA-Seq to iden- tify transcripts of ~180 nucleotides in length and longer, and small RNA-Seq to identify transcripts shorter than ~50 nucleotides. Library preparation and sequencing was com- pleted by GenomeScan (Leiden, the Netherlands) as described previously 22.
Bioinformatics analysis of small RNA and mRNA-Seq data
mRNA-Seq data was analyzed as previously described 22. For small RNA-Seq, read quality was assessed using FastQC v0.11.2 software (Babraham Institute, Cambridgeshire, UK) and Trimmomatic v0.36 was used to filter reads of low quality 23. See supplementary material for details.
Gene ontology and pathway enrichment analysis
The Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.8 (http://david-d.ncifcrf.gov) was used to test DEGs for gene ontology (GO) and path- way enrichment 24. GO terms and pathways with a Benjamini-Hochberg corrected p-value<0.05 were considered enriched. The enriched pathway list produced by DAVID was processed using Cytoscape (http://www.cytoscape.org/) to produce a visual output of the text-based pathway list 25.
Real-time quantitative PCR and ELISA
Expression of miR146a, miR147b, and the reference genes miR23a (for extracellular miRNA) and the small-nucleolar RNAs RNU6B and RNU44 (for cellular miRNA) was ana- lyzed using Taqman MicroRNA assays (Applied Biosystems, Foster City, CA, USA). cDNA was generated using Taqman MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). mRNA expression was evaluated as described previously 17, using EF1A and C1orf43 as reference genes.
Levels of IL-6 were measured in culture supernatants using the PeliKine CompactTM IL-6 ELISA kit (Sanquin, Amsterdam, the Netherlands) according to the man- ufacturer’s instructions.
Immunocytochemistry
Immunocytochemistry on cells was performed as described previously 21 using the following primary antibodies: Ki67 (clone MIB-1, monoclonal mouse, DAKO, Glostrup, Denmark, 1:200); βIII-tubulin/Tuj1 (monoclonal mouse, Neuromics, Edina, MN, USA,
 147
six



















































































   147   148   149   150   151