Computational protocol: Underexpression of HOXA11 Is Associated with Treatment Resistance and Poor Prognosis in Glioblastoma

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Protocol publication

[…] For tissue sample analysis, the total RNA was extracted from the tissue samples using the mirVana miRNA Isolation Kit (#AM1560, Ambion, Austin, TX) for microarray analysis after quantification and qualification. The total RNA quality was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The cut off RNA integrity number for RNA used in RNA amplification was 7.0 or above. The cRNA was produced using an Illumina TotalPrep RNA Amplification Kit (#IL1791, Ambion) according to the provided protocol. The cRNA was used for hybridization to a human HT12-v4 Illumina Beadchip gene expression array (Illumina, San Diego, CA) according to the manufacturer’s protocol. The arrays were scanned and the fluorescence signals were obtained using an Illumina BeadArray Reader (BeadStation 500GXDW, Illumina). The signal obtained from the scanned beadchip was transformed to intensity raw data using GenomeSortudio software (ver. 2009.1, Illumina) and was used for further data analysis. The raw data were normalized by applying a log2 transformation, quantile normalization, and gene and array centering. All data processing was performed using the R/Bioconductor packages (ver. 2.14, http://www.bioconductor.org).To determine the changes in gene expression before and after HOXA11 knockdown, the total RNA extracted from the LN18 cells transduced with siHOXA11 or control siRNA were analyzed by Affymetrix GeneChip Human Gene 1.0ST Arrays (Affymetrix, Santa Clara, CA). The RNA was amplified and labeled using a GeneChip WT Sense Target Labeling and Control Reagents Kit (Affymetrix). The cDNA was synthesized, labeled, and hybridized to the GeneChip array according to the manufacturer's protocol. The GeneChips were washed and stained using the GeneChip Fluidics Station 450 (Affymetrix), and then scanned using a GeneChip Scanner 3000 7G (Affymetrix). The expression data were normalized using the robust multi-array average method. Affymetrix Expression Console ver. 1.1 (Affymetrix) was used to compare the group signals, and the data were logtransformed (base 2) for parametric analysis. The differentially expressed genes (DEGs) were identified by significance analysis of the microarrays method in the R package ‘samr’ (R 2.11.1). [...] The data from the experiments were tested for their significance using an unpaired two-tailed Student's t test. An ANOVA and Student's t test were used to identify the significant differences in cell death rates. Kaplan-Meier curve analysis was used to generate the overall survival curves. The differences between the survival curves were analyzed using a log-rank test. The results were analyzed using IBM SPSS Statistics software ver. 19.0 (IBM Co., Armonk, NY). The data are presented as mean±standard deviation for three or more separate experiments. A p-value of 0.05 or lower was considered significant.For microarray analyses, the false discovery rates (FDRs) were calculated using three GenePattern software modules (http://www.broadinstitute.org/cancer/software/genepattern; ComparativeMarkerSelection ver. 10, HierarchicalClustering ver. 6, and HeatMapViewer ver. 13) []. The cutoff value for FDR significance was < 0.05. The significantly regulated genes were subjected to functional gene classification using the DAVID Bioinformatics Resources annotation tool (ver. 6.7, http://david.abcc.ncifcrf.gov/) []. The selected gene IDs of the identified DEGs were entered into GeneMANIA software (ver. 3.1.2.8, http://www.genemania.org) for network analysis []. […]

Pipeline specifications

Software tools Beadarray, SPSS, GenePattern, HeatMapViewer, DAVID, GeneMANIA
Applications Miscellaneous, Gene expression microarray analysis, Transcriptome data visualization
Organisms Homo sapiens
Diseases Glioblastoma, Neoplasms