Polymorphonuclear leukocytes (PMNs) were isolated from the blood of affected patients and healthy donors.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with the Qiagen RNeasy kit (or with STAT60) according to the manufacturer's instructions.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 1 ug total RNA (Expression Analysis Technical Manual, 2004, Affymetrix).
Hybridization protocol
cRNA were hybridized on Affymetrix GeneChip Human Genome U133 Plus 2.0 Array according to the standard Affymetrix protocol (Expression Analysis Technical Manual, 2004, Affymetrix). GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Scan protocol
GeneChips were scanned using an Affymetrix scanner.
Description
test PMNs
Data processing
Affymetrix GeneChip Operating Software (GCOS v1.0, http://www.affymetrix.com) was used to perform the preliminary analysis of the custom chips. All *.cel files, representing individual replicates, were scaled to a trimmed mean of 500 to produce the *.chp files. A pivot table with all samples was created including calls, call p-value and signal intensity for each gene. Statistical and quality filters were applied as follows. The average scaled expression must be at least 100 in at least one group. For probe sets more highly expressed in healthy control samples, the call must be present/marginal in at least 5 of 7 samples; for probe sets more highly expressed in test samples, the call must be present/marginal in at least 2 of 3 samples. At least a 1.5-fold change and a T test p-value of less than 0.05 are required.
