Computational protocol: New Insights into FoxE1 Functions: Identification of Direct FoxE1 Targets in Thyroid Cells

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Protocol publication

[…] FoxE1-dependent gene expression was tested using expression arrays (Agilent SurePrint Rat 60 K). We established two main comparisons: FoxE1-silenced PCCl3 cells (siFoxE1 PCCl3) vs. scrambled siRNA-treated PCCl3 cells (siScramble PCCl3) and FoxE1-silenced PCCl3 cells vs. wild type PCCL3 cells (wt PCCl3). This last condition was included to consistently analyse expression array signals in basal cellular conditions. Each comparison was performed using quadruplicates and dye swaps (Experimental design shown in ).Ten µg of total RNA for each condition were sent to the Genomics Core Unit of the Spanish National Cancer Research Centre (CNIO, Madrid) for RNA quality evaluation, amplification, labelling and hybridization to Agilent SurePrint Rat 60 K arrays according to the manufacturer’s protocols.Signal quantification was carried out with Agilent Feature Extraction Software 10.7 (Agilent Technologies, Palo Alto, CA), using default analysis parameters for Agilent’s whole rat genome 60 K gene expression arrays. To normalize the data set, we performed loess within-array normalization and quantiles between-array normalization. Differential expression analysis was done using Bioconductor’s limma package (http://www.bioconductor.org). At a later stage, we used the file “SurePrint G3 Rat GE 8×60 K Microarray” to obtain the annotations of the rat genome from Agilent. Genes that showed adjusted p-values <0.005 both in siFoxE1 vs. wild type and in siFoxE1 vs. siScramble PCCl3 cells were considered differentially expressed. Functional analysis of Gene Ontology (GO) terms was carried out using the FatiGO tool and gene set enrichment analysis was performed using FatiScan , . All microarray data can be downloaded from the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) database under accession number GSE42497. […]

Pipeline specifications

Software tools Agilent Feature Extraction, limma, Babelomics
Application Gene expression microarray analysis
Diseases Neoplasms