*2.7. Gene Expression Assay*

RNA from liver samples of the above-mentioned mice or of LSECs was extracted using the Maxwell 16 LEV simply RNA Tissue Kit (Promega, Madison, WI, USA) and the Maxwell 16 Instrument (Promega, Madison, WI, USA), according to the manufacturer's instructions.

A DNA microarray platform (Affymetrix) was used for global gene expression analysis.

Total RNA preparations were checked for RNA integrity by Agilent 2100 Bioanalyzer quality control. Mean RNA integrity number (RIN) was 7.3 ± 0.4 (range 6.5 to 8.1) for liver tissue from chow-fed animals and 7.8 ± 0.3 (range 6.5 to 8.8) for liver tissue from LCA-fed animals. RNA was further analyzed by photometric Nanodrop measurement and quantified by fluorometric Qubit RNA assays (Life Technologies).

Synthesis of biotin-labeled cDNA was performed according to the manufacturers' protocol (WT Plus Reagent Kit; Affymetrix, Inc., Thermo Fisher Scientific, Waltham, MA, USA). Briefly, 100 ng of total RNA was converted to cDNA. After amplification by in vitro transcription and 2nd cycle synthesis, cDNA was fragmented and biotin labeled by terminal transferase. Finally, end-labeled cDNA was hybridized to Affymetrix Mouse Gene 2.0 ST Gene Expression Microarrays for 16 h at 45 ◦C, stained by strepatavidin/phycoerythrin conjugate and scanned as described in the manufacturers´ protocol. Data analyses on Affymetrix CEL files are described in Section 2.14—Statistical Analysis of Expression Data. The gene expression data can be downloaded from the NCBI GEO database (https://www.ncbi.nlm.nih.gov/geo/, accession number GSE139075).

For real-time PCR, 1 μg of this RNA was used to generate cDNA utilizing the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). Gene expression was quantified using Taqman Gene Expression Assays (Thermo Fisher Scientific, Waltham, MA, USA, assay order information can be obtained upon request) and the Lightcycler 480 II (Roche Diagnostics, Rotkreuz, Switzerland). Data were produced in duplicates for each gene. Mean values of cycle numbers of the target gene were subtracted from the mean of cycle numbers of the house-keeping gene succinatdehydrogenase (SDHA) for the respective sample. These values taken to the power of 2 are the mRNA expression of the target genes in relation to SDHA expression. The number of independent experiments performed are given in the text/figure legends. At least three independent experiments were performed. In order to rule out a regulation of the chosen house-keeping gene SDHA in response to LCA feeding, we initially analyzed expression of selected genes in relation to SDHA, hypoxanthine phosphoriosyltransferase-1 (HPRT-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Since no difference in regulation was observed between these house-keeping genes, we continued the experiments with SDHA, as shown in Table 1 and Figure 3.
