Mice were kept on standard laboratory chow diet and had free access to water. Hepatic expression profiles of the four genotypes (wild type, and the three knockouts) was performed on male mice, that were all aged between 8-9 weeks.
Growth protocol
We generated mice carrying the loxP-site-flanked NEMO gene as described before (Beraza et al., 2009, J Exp Med; PMID: 19635861). From NemoΔhepa, we generated double knockout animals by crossing NemoΔhepa with constitutive TRAIL-/- and TNFR1-/- animals.
Extracted molecule
total RNA
Extraction protocol
Total RNA was purified from liver tissue using Trizol reagent (Invitrogen, Karlsruhe, Germany) andpurified using RNeasy microkit (Qiagen, Venlo, The Netherlands). RNA quality was assessed on a Agilent 2100 Bioanalyzer (Agilent Technologies, Amsterdam, The Netherlands. RNA was judged as suitable for array hybridization 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
The Ambion WT Expression kit (Life Technologies, Carlsbad, CA; P/N 4411974) in conjunction with the Affymetrix GeneChip WT Terminal Labeling kit (Affymetrix, Santa Clara, CA; P/N 900671) was used for the preparation of labeled cDNA from 100ng of total RNA without rRNA reduction.
Hybridization protocol
Hybridization of 5.5μg labelled cDNA was done overnight for 17 hours, at 60 rpm, at 45ºC in a Hybridization Oven 640 (Affymetrix). The protocol was conducted as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5). Washing and staining of the arrays were done on an Affymetrix 450 fluidics station using the protocol FS450_0001, as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5).
Scan protocol
Arrays were scanned on an Affymetrix 3000 7G scanner, as described in the Genechip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Arrays: Washing, Staining and Scanning (P/N 701028, revision 5).
Description
A202_06_TW51_939_H_(MOGENE-1_0-ST-V1).CEL
Data processing
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.14.0).
