Computational protocol: Astrocytes acquire resistance to iron-dependent oxidative stress upon proinflammatory activation

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Protocol publication

[…] After the treatments, astrocytes were subjected to RNA extraction using Trizol (Invitrogen), according to the manufacturer’s instructions. The gene expression profiling was determined using RatRef-12 Expression Beadchips (Illumina Inc., San Diego, CA, USA). Each beadchip investigates for more than 21,000 transcripts selected primarily from the NCBI RefSeq database (Release 16) and in a minor part from the UniGene database. An amount of 500 ng of total RNA was reverse transcribed into complementary RNA and biotin-UTP labeled using the Illumina TotalPrep RNA Amplification Kit (Applied Biosystems) according to the manufacturer’s protocol; 750 ng of complementary RNA was then hybridized to the BeadChip array and stained with streptavidin-Cy3. All procedures were performed following the manufacturer’s instructions. BeadChips were imaged using the Illumina BeadArray Reader, a two-channel 0.8-μm-resolution confocal laser scanner and Illumina BeadScan software. Illumina GenomeStudio v.2011.1 software was used to elaborate the fluorescence signal to a value whose intensity corresponded to the quantity of the respective transcript in the original sample. The same software was used to assess the quality controls, which included the biological specimen controls, hybridization controls, signal generation controls and negative controls. The samples belonging to three different experimental conditions (i.e., resting astrocytes, astrocytes stimulated with cytokines and astrocytes stimulated with microglia-conditioned medium) were tested in technical duplicates, and a scatter plot with a correlation coefficient for each couple of replicates was calculated, leading to a mean value of 0.99. The fold change based on raw data was calculated for the comparisons.Data analysis was performed using the Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems Inc., Redwood City, CA, USA). lllumina probes, filtered for p < 0.05 and a threshold of 1.5 points of fold change between different conditions (in upregulation or downregulation) were uploaded into the software together with their differential expression p-values measured by t-test. Each probe was mapped to its own gene object in the Ingenuity Pathways Knowledge Database.To interpret the gene expression results of the different condition of stimulation in the context of biological processes, pathways and networks, IPA Core Analyses were conducted. Networks of these genes were assigned a score based on their connectivity: the score reflected the number of focus genes in the network and how relevant this network is to the original list of focus genes. The significance of the association between the data set of genes and the canonical pathways contained in the Ingenuity Pathways Knowledge Database was determined by adjusted p-values using the Benjamini-Hochberg correction for multiple testing, and a p-value < 0.05 after correction was considered statistically significant according to functional enrichment analysis. Besides, the “overlay” function was used to give a graphic visualization of which subset of genes inside a given canonical pathway was modulated in different conditions. […]

Pipeline specifications

Software tools Beadarray, GenomeStudio, IPA
Application Gene expression microarray analysis
Diseases Neoplasms, Iron Overload
Chemicals Iron