Pancreata were dissected from 12.5, 14.5 and 16.5 day mouse embryos taken from timed matings of Pdx1Cre/+ ; beta-cateninflox/+ mice and immediately submerged in RNAlater [Ambion, Inc., Austin, TX]. Quality control steps: Total RNA was purified using RNeasy columns [Qiagen, Valencia, CA] according to the manufacturer’s directions and quality was assessed using an Agilent 2100 Bioanalyzer [Agilent Technologies, Inc., Palo Alto, CA].
Label
biotin
Label protocol
120 ng of total RNA from 2 wild type [WT] and 4 beta-catenin knock-out [KO] pancreata at the 14.5 and 16.5 time points was amplified twice with the Arcturus RiboAmp kit [Arcturus Bioscience, Inc., Mountain View, CA] and cRNA was labeled with biotin-UTP, CTP using T7 RNA polymerase [Enzo Life Sciences, Inc., Farmingdale, NY].
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), 0.1 mg/mL Herring Sperm DNA (Promega), 0.5 mg/mL Acetylated BSA (Invitrogen), and 1X Hybridization Buffer. Heat hybridization cocktail to 99oC for 5 minutes, to 45oC for 5 minutes, and spin at maximum speed in a microcentrifuge for 5 minutes. Fill probe array with 200 ?L of 1X Hybridization Buffer. Incubate at 45oC for 10 minutes in the GeneChip Hybridization Oven 640 (Affymetrix) rotating at 60 rpm. Remove 1X Hybridization Buffer and fill probe array with 200 ?L of the hybridization cocktail. Incubate at 45oC 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 EukGE-WS2v5_450. 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
Wild type [WT] pancreata were dissected from day 14 and 16 mouse embryos. A Pdx1-cre transgenic line was crossed with a floxed allel of beta-catenin to generate beta-catenin knock-out [KO] pancreata at the 14.5 and 16.5 time points.
Data processing
Affymetrix murine MOE 430 2.0 gene chips were used.GeneChip CEL files were subjected to RMA normalization using the GeneSpring GX 7.1. 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 7.1 (Agilent technologies Inc. Palo Alto, California) was used to normalization, Clustering and filtering. Individual clusters were then analyzed for functional relatedness of genes within the cluster using David 2.0(http://david.abcc.ncifcrf.gov/summary.jsp).
