Description: <strong>Idizationsfrom total RNA with the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26748734 Affymetrix 3'IVT Express Kit (Affymetrix, Santa</strong><blockquote> </blockquote><blockquote>Idizationsfrom total RNA with the Affymetrix 3'IVT Express Kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer's instructions with an input of 100 ng total RNA. The Agilent Bioanalyzer (Agilent, Amstelveen, the Netherlands) and the Shimadzu MultiNA Bioanalyzer (Shimadzu,Tokyo, Japan) were used to examine the quality of cRNA to confirm that the average <a href="https://www.medchemexpress.com/Dovitinib.html">Dovitinib</a> fragment size was according to Affymetrix' specifications. For each sample, 7.5ug biotinylated cRNA was fragmented and hybridized in a final concentration of 0.0375 ug /ul on the Affymetrix HT Mouse genome 430 PM array (Affymetrix, Santa Clara, CA, USA). After washing and staining by the GeneTitan instrument (Affymetrix, Santa Clara, CA, USA) using the Affymetrix HWS kit for Gene Titan, absolute values of expression were calculated from the scanned array using the Affymetrix Command Console v 3.2 software. The data Quality Control was performed using the Affymetrix Expression Console v 1.1 (Affymetrix, Santa Clara, CA, USA) software to check whether all parameters met the quality specifications. The Probe Logarithmic Intensity Error Estimation (PLIER) algorithm method was used for probe summarisation [26]. In order to monitor the sample independent control and the performance of each individual sample during hybridization, hybridization controls were added to the hybridization mixture. To determine the biological variation between samples, the sample dependent controls such as internal control genes, background values, and average signals were used. In summary, all the data were within data Quality Control thresholds, according to the Affymetrix Expression Console specifications. Nonnormalized data in a form of the Cell Intensity File (*.CEL) were re-annotated (EntrezGene htmg430pm_ Mm_ENTREZG) and the data were RMA normalized [26]. The microarray data generated in this study were deposited <a href="https://www.ncbi.nlm.nih.gov/pubmed/28501505" title="View">PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28501505</a> to gene expression omnibus (GEO) and the GEO accession number is GSE63457 (data will be public upon acceptance of the manuscript).Identification of significantly affected genesGene expression analysis in PCLS incubated for 24 h with CsA or DMSO was performed using the HT Mouse Genome 430 PM array plate (s) using the Affymetrix GeneTitan system (Affymetrix, Santa Clara, CA, USA). RNA was extracted from co-cultured slices using the RNeasy Tissue Mini Kit (Qiagen, Venlo, The Netherlands) according to the manufacturer's protocol. RNA concentration and purity were assessed using a NanoDrop ND-1000 spectrophotometer (Isogen IJsselstein, The Netherlands) by measuring absorption ratios at 260/280 and 230/ 280 nm. The integrity of the RNA samples was checked using the Shimadzu MultiNA Bioanalyzer (Shimadzu, Tokyo, Japan). Biotin- labelled cRNA was generatedTo identify genes significantly affected by CsA in WTPCLS and FXRKO-PCLS, the normalized data were log2 transformed, followed by analysis of variance (ANOVA) with Benjamini-Hochberg correction for false discovery rate (FDR). Genes were considered significant if FDR < = 0.05 and fold change (FC) was or 1.5 for down-or upregulated genes respectively.Data mining Pathway analysis (MetaCore)MetaCore identifies pathways using a default enrichment analysis. Genes significantly affected by CsA in WT-PCLS and FXRKO-PCLS (FDR 0.05) were uploaded to MetaCore for pathway analysis. The pathway analysisSzalowska et al. BMC Genomics (2015) 16:Page 5 ofwas performed using Functional Ontology Enrichment/ Pathways Maps default option in MetaCore for mouse. Th.</blockquote>