Computational protocol: Guanabenz Prevents d-Galactosamine/Lipopolysaccharide-Induced Liver Damage and Mortality

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Protocol publication

[…] Total RNA was isolated with RNeasy kit (Qiagen). cDNA was synthesized with random hexamers and superscript II reverse transcriptase (Invitrogen). For Affymetrix microarray analysis, GM-CSF DCs were cultured in RPMI supplemented with 5% heat-inactivated fetal bovine serum, 50 µM beta-mercaptoethanol and GM-CSF. Cells were differentiated for 6 days before the experiment. Cells were treated for 8 h with Proteus mirabilis and harvested before lysis. Control and P. mirabilis treated DCs were incubated with the bacteriostatic chloramphenicol to avoid bacterial overgrowth. GBZ was used at 50 µM and added at the same time as P. mirabilis. Each condition was analyzed in independent triplicates (n = 3). Hybridization to arrays (Affymetrix GeneChip Mouse Gene 1.0 ST) and image scanning were performed according to the Affymetrix Expression Analysis Technical Manual. Gene Expression microarray raw data were normalized using limmaGUI software (R/Bioconductor, Boston, MA, USA). Data can be accessed through the GEO repository accession number GSE90831. Microarrays data were analyzed with the ingenuity pathway analysis (IPA) software (Qiagen). Quantitative real time PCR analysis was performed with Applied Biosystems PRISM 7700 Sequence Detection System. The primers used are: mouse IL-10 forward: 5′-CAGCAGACTCAATACACACT-3′; mouse IL-10 reverse: 5′-TGGCCCAGAAATCAAGGAGC-3′; mouse RPS16 (housekeeping) forward: 5′-CTTGGATTCATCCACATA-3′; mouse RPS16 reverse: 5′-ATTTGCTGGTGTGGATATTCG-3′. […]

Pipeline specifications

Software tools limmaGUI, IPA
Application Gene expression microarray analysis
Organisms Mus musculus
Diseases Liver Diseases, Neoplasms, Liver Failure, Acute
Chemicals Lipopolysaccharides