Computational protocol: Hypoxia-inducible C-to-U coding RNA editing downregulates SDHB in monocytes

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Protocol publication

[…] Expression profiling was accomplished using the Human HT-12 whole-genome gene expression array and direct hybridization assay (Illumina, Inc.). Initially, 500 ng total RNA was converted to cDNA, followed by in vitro transcription to generate biotin labeled cRNA using the Ambion Illumina TotalPrep RNA Amplification Kit (Ambion, Inc.) as per manufacturer’s instructions. 750 ng of the labeled probes were then mixed with hybridization reagents and hybridized overnight at 58°C to the HT-12v4 BeadChips. Following washing and staining with Cy3-streptavidin conjugate, the BeadChips were imaged using the Illumina iScan Reader to measure fluorescence intensity at each probe. The intensity of the signal corresponds to the quantity of the respective mRNA in the original sample.The background corrected gene expression levels were extracted from BeadChip using Illumina’s Genome Studio (v2011.1) gene expression module (v1.9.0). The log2 transformed expression levels were quantile normalized using Lumi module in the R-based Bioconductor package (). For data quality control, we excluded the genes with detection p-value greater than 0.05 (i.e., indistinguishable from the background noise). 18941 out of 34686 genes passed this filtering for downstream analysis.The Limma program () was used to calculate the level of differential gene expression. Briefly, a linear model was fit to the data (paired design, with cell means corresponding to the different condition and a random effect for array) and selected contrasts of condition (i.e., case vs. control) were performed. A list of differentially expressed genes with P < 0.05 and ≥ 2 fold-change was obtained and analyzed for enriched Gene Ontology (GO) categories and KEGG pathways using NCBI DAVID Bioinformatics Resources (). The enriched GO terms and KEGG pathways with P < 0.05 and ≥ 5 genes were kept. […]

Pipeline specifications

Software tools limma, DAVID
Databases KEGG
Application Gene expression microarray analysis
Organisms Homo sapiens
Diseases Neoplasms
Chemicals Oxygen, Succinic Acid