|Public on Sep 19, 2012
|background strain: BalbC
|FSAP-/- mice were backcrossed on to the BalbC background up to the F3 generation. FSAP-/- and littermate FSAP+/+ (WT) mice obtained from crossings between heterozygous mice were used for the experiment between 20 and 30 weeks of age. Surgical ligation of the common bile duct (BDL) was performed under isoflurane anesthesia and sham-operated mice underwent the same procedures except that the bile duct was not ligated. After 2 weeks the mice were sacrificed and the liver was flash frozen and stored at -80°C for further analysis.
|Liver RNA was isolated by guanidine thiocyanate, chloroform and isopropanol precipitation, followed by DNAase digestion. Quantification and quality control of the RNA was checked with Agilent 2100 Bioanalyzer platform (Agilent Technologies, Palo Alto, CA, USA). Only total RNA with an RNA integrity number ≥ 7.2 was used for microarray hybridization (N = 3 mice per group)
|100 ng of each total RNA samples was used for the amplification and labeling step using the Agilent Quick Amp Labeling Kit (Agilent Technologies). Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies).
|The hybridization procedure was performed according to the the “One-Color Microarray-Based Gene Expression Analysis protocol (version 5.7, part number G4140-90040) using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, 1.65 µg Cy3-labeled fragmented cRNA in hybridization buffer was hybridized overnight (17 hours, 65 °C) to Agilent Whole Mouse Genome Oligo Microarrays 4x44K using Agilent’s recommended hybridization chamber and oven. Following hybridization, the microarrays were washed once with the Agilent Gene Expression Wash Buffer 1 for 1 min at room temperature followed by a second wash with preheated Agilent Gene Expression Wash Buffer 2 (37 °C) for 1 min. The last washing step was performed with acetonitrile.
|Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (Agilent Technologies). The Agilent Feature Extraction Software 9.1 was used to read out and process the microarray image files.
|FES derived output data files were further analyzed using the Rosetta Resolver gene expression data analysis system (Rosetta Biosoftware, Rosetta error model; Lee Weng et al 2006).
|Feb 24, 2012
|Last update date
|Sep 19, 2012
|Miltenyi Biotec GmbH
|Factor VII activating protease (FSAP) exerts antifibrotic effects in a mouse model of experimental liver fibrosis