Lysis matrix b tubes

Trim24 genotyping was performed on genomic DNA obtained from tail biopsy sampled on 10-day-old mice, by means of a standard PCR technique, using Chromo 4 Thermocycler (Bio-rad, Marnes-la-Coquette, France). Primers sequences are available in Additional file 2.
Gene expression analysis in tumor and normal liver samples was performed using the RT q-PCR technique. Tissue samples were homogenized in a FastPrep®-24 Instrument (MP Biomedicals Inc., lllkirch, France) in Lysis Matrix B tubes (MP Biomedicals, Inc.) containing lysis buffer (Sigma-Aldrich, Saint Quentin Fallavier, France). RNAs were then purified with the mammalian GenElute Gel Extraction Kit (Sigma-Aldrich), according to the manufacturer’s recommendations. RNAs (3 μg) were used as template for reverse transcription with random hexamer and anchored oligo dT, in the presence of 200 units of reverse transcriptase (MP Biomedicals, Inc.). Resulting cDNAs were analyzed using the RT qPCR on a Chromo 4 Cycler, using QuanTitect MasterMix (Qiagen, Courtaboeuf, France) and the primers corresponding to genes of interest (see Additional file 2). Results were analyzed with the Opticom 3 software (Bio-rad). Expressions of genes of interest were normalized by housekeeping gene HPRT and represented as tumor/liver ratio.

Tumor and normal liver samples were homogenized in a Fastprep apparatus (MP Biomedical) in Lysis Matrix B tubes (MP Biomedical) containing lysis buffer (Sigma). RNAs were purified with the mammalian Genelute kit from Sigma, according to the manufacturer recommendations. 3 µg of RNA were used as template for reverse transcription with random hexamer in the presence of 200 units of Superscript II reverse transcriptase (Invitrogen). cDNA were analyzed by real time quantitative PCR on a Chromo4 (Biorad), using Quantitect mastermix (Qiagen). Primer sequences for the PCR were as follows: 36B4: 5′-GAGGTCACTGTGCCAGCTCA-3′ and 5′-GAAGGTGTACTCAGTCTCCA-3′; AFP: 5′-GGCAAAGCCCTACAGACCA-3′ and 5′-TAAACGCCCAAAGCATCAC-3′; ALB: 5′-CCCTGTTGCTGAGACTTGCT-3′ and 5′-CTGAGGTGCTTTCTGGGTGT-3′; ASMA: 5′-GGCTGTGCTGTCCCTCTATG-3′ and 5′-TCTCACGCTCGGCAGTAGTC-3′; CCND1: 5′-AACTACCTGGACCGCTTCCT-3′ and 5′-GCTTGTTCTCATCCGCCTCT-3′; COLA1: 5′-TTTGGAGAGAGCATGACCGA-3′ and 5′-AAGTTCCGGTGTGACTCGTG-3′; IL-6: 5′-AGGATACCACTCCCAACAGAC-3′ and 5′-AGTGCATCATCGTTGTTCATAC-3′; FN1: 5′-CCACCTCGAGCCCGTTATAG-3′ and 5′-GCAGAGGCTGCAGGGTAGTA-3′; MMP3: 5′-CACGAGGAGCTAGCAGGTTA-3′ and 5′-TCCAACTGCGAAGATCCACT-3′; PDGFB: 5′-CCGGTCCAGGTGAGAAAGAT-3′ and 5′-AATAACCCTGCCCACACTCT-3′; SPP1: 5′-GCTTGGCTTATGGACTGAGG-3′ and 5′-CTCTCCTGGCTCTCTTTGGA-3′; TGFB: 5′-ATTCAGCGCTCACTGCTCTT-3′ and 5′-ACTTCCAACCCAGGTCCTTC-3′.