Whole blood from normal children and children with SIRS, sepsis, or septic shock.
Treatment protocol
Samples were not manipulated.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from whole blood using the PaxGene protocol. 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
Create a hybridization cocktail for a single probe array that contains 0.05 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 99°C for 5 minutes, to 45°C 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 45°C 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 45°C 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]
Commercial Affymetrix Human Genome U133 Plus 2.0 GeneChip (Affymetrix, Santa Clara, CA). 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 by the signals of the 3' probe set to the 5' probe set of the internal control genes, GAPDH and beta-actin, with acceptable 3' to 5' ratios between 1 and 3. Eukaryotic Spike controls were used to determine that the hybridization of target RNA to the array occurred properly. GeneSpring 7.2 (Agilent technologies Inc. Palo Alto, California) was used for normalization, clustering, filtering, and statistical analyses. The Raw CEL files were processed using the RMA (Robust Multichip Average) built in GeneSpring software. All the samples were then normalized to the median of the controls.
