Genechip ht 3 ivt express kit

Five mice in each group were left untreated (naive group) or adoptively transferred with MOG-reactive Th1 or Th17 cells. Act1 KO mice received only the Th17 adoptive transfer. Twelve days after adoptive transfer, the mice were killed, and spinal cords were subjected to mRNA extraction for microarray analysis. Target preparation was performed on a Biomek FXP (Beckman Coulter, Brea, CA) using a GeneChip HT 3′IVT Express Kit (Affymetrix, Santa Clara, CA) in accordance with the manufacturer's instruction. Labelled cRNA was hybridized on an Affymetrix GeneChip HT-MG-430PM-96 (Affymetrix). Array hybridization, washing and scanning were performed using GeneTitan (Affymetrix). Three independent biological replicates were analysed in each experiment, which yielded consistent results. The t-test was used to assess significance, and P<0.05 was deemed significant. The data for the normalized GeneChip analysis are provided in Supplementary Data 1 and the raw data are deposited in the Gene Expression Omnibus.

Cytoplasmic and nuclear RNA were extracted from WT and IRAK2-deficient macrophages and subjected to microarray analysis. Targets preparation was performed on a Biomek FXP (Beckman Coulter) using a GeneChip HT 3′IVT Express Kit (Affymetrix, Santa Clara, CA) in accordance with the manufacturer’s instruction. Labeled cRNA were hybridized on an Affymetrix GeneChip HT-MG-430PM-96 (Affymetrix). Array hybridization, washing, and scanning were performed on GeneTitan (Affymetrix). Three independent biological replicates were analyzed in each experiment. Probe signals were subjected to presence calling and normalized to derive relative transcript abundance. The ratio (R) between the nuclear (N) and cytoplasmic (C) abundance of each transcript is calculated (R = N/C) to quantify the nuclear retention of the transcript. The nuclear/cytoplasmic ratio of the transcript in the LPS-treated IRAK2 knockout BMDMs (RKO) is then divided by that of the same transcript in the corresponding wild-type BMDMs (RWT). The resulting value is used as an index (I = RKO/ RWT) to measure the impact of IRAK2 deficiency on the nuclear retention of the transcript. Transcripts induced by LPS treatment are ranked based on the index I and the top 70 transcripts are used as the positive set for motif enrichment analysis.

A microarray analysis was initially performed for all samples (74) to detect differentially expressed genes between groups of interest. For expression profiling, 100 ng of total RNA was isolated, linearly amplified, and biotinylated with the GeneChip^® HT 3′IVT Express Kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. Fifteen micrograms of labeled and fragmented cRNA were then hybridized onto Human Genome U219 Gene Chip^® arrays (Affymetrix). Hybridization, washing, staining and scanning were automatically performed in a GeneTitan^TM instrument (Affymetrix). Scanned images were subjected to visual inspection to control for hybridization artifacts and proper grid alignment, and they were analyzed with AGCC 3.0 (Affymetrix) to generate CEL files.