Affymetrix expression console software

Gene expression data of human tumor and normal tissues were obtained from publicly available datasets including, the Gene Expression Omnibus (GEO) GSE66533, GSE87437, GSE141690. Gene expression and sarcoma patient clinical data was obtained from the Innovative Therapies for Children with Cancer consortium cohort (rhabdomyosarcoma), the GEO dataset GSE21257 (osteosarcoma), and the adult sarcoma cohort from The Cancer Genome Atlas program (TCGA‐SARC). The RNA raw data were normalized and compared using the Affymetrix Expression Console Software (Thermofisher). Pathway analysis of differentially expressed genes was conducted by Metascape.^[45]

The RNA expression arrays method has been previously described^{20 (link)}. Briefly, RNA from each sample was used to generate amplified and biotinylated sense-strand cDNA from the entire expressed genome according to the Sensation Plus FFPE Amplification and WT Labeling Kit (P/N 703089, Rev.4 Thermo Fisher Scientific Inc., Life Technologies). GeneChip ST Arrays (GeneChip Human Gene 2.1 ST Array Plate) were hybridized, washed, stained, and finally scanned with the GeneTitan Multichannel (MC) Instrument, according to the GeneTitan Instrument User Guide for Expression Array Plates (PN 702933, Thermo Fisher, Scientific Inc., Life Technologies).
The RNA raw data were normalized and compared using the free Affymetrix Expression Console Software provided by Thermo Fisher. Pathway analysis of differentially expressed genes was conducted by Metascape⁴¹ .

Expression data from triplicate samples were generated by Affymetrix Expression Console software (version 1.1, Affymetrix). Normalization was performed using the robust multiaverage (RMA) algorithm implemented in Affymetrix Expression Console software. Whether genes were differentially expressed among the three groups was determined using a one-way ANOVA of the RMA expression values. A multiple testing correction was applied to the p values of the F statistics to adjust the false discovery rate[16] (link). Genes with adjusted F-statistic p values of <0.05 were extracted. Genes with significantly differences in expression between the 2 groups, with a greater-than twofold difference between the control and each test group were selected for the further study.
Genes with similar expression patterns, co-expressed by both groups were identified through hierarchical clustering and K-mean clustering using MultiExperiment Viewer software version 4.4 (www.tm4.org, Dana-Farber Cancer Institute, MA, USA). A Web-based tool, the Database for Annotation, Visualization, and Integrated Discovery (DAVID), was used to assess the biological interpretation of differentially expressed genes. These genes were then classified based on the gene function in the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway database (http://david.abcc.ncifcrf.gov/home.jsp).

Total RNA was extracted from BLT1^hi and BLT1^lo DCs (treated with LPS or CpG DNA or left untreated for 4 h) using TRIzol reagents. cDNA was prepared and labeled using the Ambion WT Expression Kit (Affymetrix, San Diego, CA, USA) and GeneChip WT Terminal Labeling Kit (Affymetrix). The labeled samples were subjected to hybridization with the GeneChip Hybridization Wash, Stain Kit (Affymetrix) and GeneChip Mouse Gene 1.0 ST array (Affymetrix). Signals were scanned with a GeneChip Scanner 3000 7 G, and data were analyzed using Affymetrix Expression Console software (Affymetrix). The robust multiarray average algorithm was used for log2 transformation and normalization of the GeneChip data. Hierarchical clustering analysis was performed using R (www.r-project.org). Functional enrichment analysis of selected genes was performed based on GO pathway annotation terms, with P values < 0.05 considered statistically significant.

The Affymetrix Genechip Mouse Clariom S was used to examine gene expression (Affymetrix, Santa Clara, CA, USA). Sample processing and generation of Microarray data was completed by experienced technicians in the Molecular Resource Center at UTHSC. In brief, two hundred nanograms of DNase-treated total RNA was amplified, labeled, and fragmented using Ambion Whole Transcript (WT) Expression Kit according to the manufacturer’s protocol (Thermo Fisher Scientific, Santa Clara, CA USA). Samples were hybridized overnight according to manufacturer’s protocols; samples were then washed and stained on Affymetrix GeneChip Fluidics Station 450 (Affymetrix, Santa Clara, CA, USA) followed by scanning on the GeneChip Scanner 3000 (Applied Biosystems, Waltham, MA, USA). Data was normalized and analyzed for quality control in Affymetrix Expression Console Software using RMA-sketch normalization (Affymetrix, Santa Clara, CA, USA). After normalization and quality control, a total number of 22,203 probe sets were used for subsequent data analysis. A total of 128 samples were used—4 samples per treatment (control, ethanol), per sex (male, female), and per strain (B6, D2, BXD2, BXD48a, BXD60, BXD71, BXD73, BXD100) (Supplemental Table S1).