Mice had free access to chow and water. At the indicated time points, mice were killed and livers were removed. Livers were macroscopically dissected and tumor material, non-tumorous control liver tissue as well as liver tissue from untreated, sex- and age-matched control mice were immediately snap frozen, followed by histopathological confirmation of the tumor tissue.
Growth protocol
Diethylnitrosamine (DEN) driven liver tumors [PMID: 15329734], Lymphotoxin-α/b-(LTa/b) driven tumors [PMID: 15661974] and Myc-driven liver tumors [PMID: 17589519] were generated as described previously. In brief, for generation of DEN-driven tumors, mice were given intraperitoneal injections of DEN (Sigma) at a dose of 10 mg per kg body weight at 15 d of age [PMID: 26502405]. Mice were observed for development of tumors at 9 months of age. For LTα/β driven tumors, tg1223 mice expressing LTα and -β in a liver-specific manner at high level were followed for 12 months for tumor development [PMID: 19800575]. Finally, for Myc-driven liver tumors, TRE-MYC mice were crossed to LAP-tTA (liver-specific promoter) mice. Animals were maintained on doxycycline (200 mg/kg doxy chow) to suppress MYC expression until 8 weeks of age. Doxycycline was then removed, and mice were followed for evidence of tumor formation [PMID: 19061838].
Extracted molecule
total RNA
Extraction protocol
Total RNA, along with miRNA, was isolated by Directzol RNA Miniprep (Zymo Research, Irvine, CA). RNA integrity was checked on chip analysis (Agilent 2100 Bioanalyzer, Agilent Technologies, Amsterdam, The Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for analysis only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0).
Label
biotin
Label protocol
Purified total RNA (100ng per sample) was labeled with the Whole-Transcript Sense Target Assay (Affymetrix, Santa Clara, CA, USA; P/N 900652).
Hybridization protocol
Hybridization and washing of the Affymetrix GeneChip Mouse Gene 1.1 ST peg arrays were performed on a GeneTitan Instrument (Affymetrix, Santa Clara, CA) according to the manufacturer's recommendations.
Scan protocol
Arrays were scanned on an Affymetrix GeneTitan instrument (Affymetrix, Santa Clara, CA).
Data processing
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.40.2).
