Total RNA was extracted from whole blood samples using the PaxGene Blood RNA System (PreAnalytiX, Qiagen/Becton Dickson) according the manufacturer’s specifications. 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
Labeling involved an in vitro transcription reaction using the ENZO BioArray HighYield RNA Transcript Labeling Kit (Affymetrix) according to manufacturer’s instructions.
Hybridization protocol
The protocol and conditions used during hybridization, blocking and washing: 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
Scan protocol
The image file was captured on an Affymetrix Gene Chip Scanner 3000
Description
Experimental factor value: Day 1 samples = control, SIRS, or septic shock. Day 3 samples = SIRS, septic shock, or SIRS resolved
Data processing
Processed with Microarray suite 5.0 (Affymetrix) to generate .CEL files that were subject to RMA normalization (Irizarry et al 2003) using GeneSpring software. 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 7.2 (Agilent technologies Inc. Palo Alto, California) was used to normalization, Clustering and filtering. 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
