Eukge ws2v5 450

Sorted IL-17+ cell samples were lysed in 500 μl of TRIzol (Invitrogen). Chloroform (0.2 ml) was added to 1 ml TRIzol cell homogenate and whirl mixed for 15 sec. Homogenates were incubated 2-3 min at RT and centrifuged 15 min at 10,000g (4°C). The water phase containing the RNA was further purified using RNeasy MinElute Cleanup Kit (Qiagen). RNA integrity was confirmed on an Agilent 2100 Bioanalyzer using total RNA nano chips (Agilent Technologies, Santa Clara, CA, USA). An amount of 100 ng of total RNA was used to prepare targets by 3′ IVT Express kit (Affymetrix, Santa Clara, CA, USA) following manufacturer’s instructions. Hybridization cocktails were hybridized onto Human Genome U133 Plus 2.0 Gene Chips® (Affymetrix) at 45°C for 17 h (60 rpm) in a Hybridization Oven 640 (Affymetrix). GeneChips® were washed and stained in a GeneChip® fluidics station 450 using the fluidics protocol “EukGE-WS2v5_450” (Affymetrix). Chips were scanned in a GeneChip® scanner 3000 (Affymetrix). Microarray data were normalized and gene expression measures derived using the RMA algorithm and the Bioconductor package “Affy” (http://www.bioconductor.org). Custom CDF (chip definition file) from brainarray.mbni.med.umich.edu was used. Qlucore Omics Explorer 2.2 (Qlucore AB, Sweden) was used for the statistical analysis of the normalized data.