Total RNA was extracted from the murine spleens using Trizol kit (Invitrogen) and then purified two times using RNeasy columns (Qiagen) Quality control steps: RNA quality was assessed by using the Agilent bioanalyzer (Agilent Technologies, Palo Alto, CA) and only those samples with 28S/18S ratios between 1.3 and 2 were subsequently used
Label
biotin
Label protocol
Standard Affymetrix one-cycle target labeling protocol.
Hybridization protocol
Full Protocol Description: Create a hybridization cocktail for a single probe array that contains 0.067 ug/uL fragmented cRNA, 50 pM Control Oligonucleotide B2 (Affymetrix), 20X Eukaryotic Hybridization Controls (1.5 pM bioB, 5 pM bioC, 25 pM bioD, 100 pM cre) (Affymetrix), 10% DMSO, and 1X Hybridization Buffer. Heat hybridization cocktail to 99C for 5 minutes, to 45C for 5 minutes, and spin at maximum speed in a microcentrifuge for 5 minutes. Fill probe array with 200 uL of 1X Hybridization Buffer. Incubate at 45C for 10 minutes in the GeneChip Hybridization Oven 640 (Affymetrix) rotating at 60 rpm. Remove 1X Hybridization Buffer and fill probe array with 200 uL of the hybridization cocktail. Incubate at 45C for 16 hrs in the Hybridization Oven rotating at 60 rpm. Fluidics: Wash and Stain probe arrays using the Fluidics Station 450 (Affymetrix) utilizing the fluidics protocol FS450-0004. Arrays were stained with phycoerythrin-conjugated streptavidin [Molecular Probes, Eugene, OR] and hybridization signals were amplified using antibody amplification with goat IgG [Sigma-Aldrich] and anti-streptavidin biotinylated antibody [Vector Laboratories, Burlingame, CA], as described in the Affymetrix GeneChip® Expression Analysis Manual
Scan protocol
Images were scanned using a Genechip scanner 3000 [Affymetrix]
Description
Samples used for hybridization consisted of non-pooled (NP) RNA extracts from the two genotypic groups namely wild type controls (WT) and Nix knockout animals (Nix-/-). A total of eight hybridization experiments were performed.
Data processing
Affymetrix murine MOE 430 2.0 gene chips were used.GeneChip CEL files were subjected to GC-RMA normalization (get reference) using the GeneSpring GX 7.3.1 Standard Affymetrix internal control genes were used to check the quality of the assay quality by the signals of the 3' probe set to the 5' probe set of the internal control genes, GAPDH and B-actin, with acceptable 3' to 5' ratios between1-3. Prokaryotic Spike controls were used to determine the hybridization of target RNA to the array occurred properly. GeneSpring 7.3.1 (Agilent technologies Inc. Palo Alto, California) was used to normalization, Clustering and filtering.
