Genechip rat gene 1.0 st array

Total RNA was extracted from frozen liver samples with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Equal amounts of RNA from 3 individual livers were combined and 10 μg of RNA was used for biotin-labeled complementary RNA (cRNA). Labeled and fragmented cRNA was subsequently hybridized to the GeneChip Rat Gene 1.0 ST Array (Affymetrix, Santa Clara, CA, USA). Labeling, hybridization, image scanning and data analysis were performed at Kurabo Industries Ltd. (Osaka, Japan).

For exosome mRNA analysis, total RNA (n = 3) was isolated from exosomal pellets using the RNeasy Mini Kit including the DNase digestion step (Qiagen, Hilden, Germany). Subsequently, two rounds of amplifications were carried out with the Riboamp HS Amplification Kit according to the manufacturer's instructions. RNA quantity was assessed using the NanoDrop 1000 (Thermo Scientific Nano Drop Technologies, Wilmington, DE, USA). Probes for the GeneChip RatGene 1.0 ST Array (each 300 ng) were prepared according to the Ambion WT Expression Kit (Ambion, Kaufungen, Germany) and the Affymetrix GeneChip WT Terminal Labeling Kit (Affymetrix, Santa Clara, CA, USA) manuals. The fragmented labeled sample was hybridized to an Affymetrix GeneChip RatGene 1.0 ST Array at 45°C for 16 hours at 60 rpm. Hybridised arrays were then washed and stained on Fluidics Station 450 (protocol FS_450_00007) and scanned on a GeneChip Scanner 3000 7G (both Affymetrix). The image data were analyzed with GeneChip Command Console Software (Affymetrix). Cell intensity files were processed by robust multiarray averaging [12] (link) using Affymetrix Power Tools, distributed through AltAnalyze [13] , using constitutive probe sets with detection above background p-values <0.01 and a raw expression threshold of 50. Threefold changes with a p-value of ≤0.01 were used as cut-off for up/down regulation.

Hepatocytes were incubated in the presence of 0.1 nM IL-1β with or without 100 μg/ml FRLFE for 2.5 h, and total RNA was purified using an RNAqueous kit (Applied Biosystems). Total RNA was labeled using an Ambion WT Expression Kit (Affymetrix Inc., Santa Clara, CA, USA) and a GeneChip WT Terminal Labeling and Controls Kit (Affymetrix Inc.) and was subjected to expression analysis using the GeneChip Rat Gene 1.0 ST Array (Affymetrix Inc.). The expression data were analyzed using the Expression Console Software (Affymetrix Inc.). Significant changes in mRNA expression were predicted by the signal ratios and Z score transformation [36] (link). To determine the ‘increased transcripts’ in the hepatocytes treated with FRLFE and IL-1β compared to the hepatocytes treated with IL-1β alone, we selected probe sets with a signal ratio ≥2.0 and a Z score ≥2.0. For the ‘decreased transcripts’ caused by FRLFE treatment, we selected probe sets with a signal ratio ≤0.5 and a Z score ≤−2.0.