Total RNA was extracted from whole blood using the PaxGene system. 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
Labeling involved an in vitro transcription reaction using the ENZO BioArray HighYield RNA Transcript Labeling Kit (Affymetrix) according to manufacturer's instructions.
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]
Description
1) Control day 1; 2) Septic shock day 1; 3) Septic shock day 3 (same patient cohort as Septic shock day 1).
Data processing
The image file was captured on an Affymetrix GeneChip Scanner 3000 and initially processed with Microarray suite 5.0 (Affymetrix). 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. Each array was normalized the same. Global scaling is used by adjusting the average intensity or signal value of each probe array to the same Target Intensity Value (TGV) of 1500. Genechip Operating Software 1v4 (Affymetrix) was then used to generate CEL files from each experiment, which were then imported into GeneSpring for all further data analysis. 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.
