Computational protocol: Genome-wide analysis of Pax8 binding provides new insights into thyroid functions

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Protocol publication

[…] The Pax8-dependent gene expression study was performed in differentiated PCCl3 thyroid cells by means of expression arrays (Agilent rat whole genome 44 K arrays). For this purpose, we generated three different conditions to finally establish two main comparisons: wild type vs. Pax8-silenced PCCl3 cells (siPax8 PCCl3), and scrambled siRNA-treated (siScramble PCCl3) vs. Pax8-silenced PCCl3 cells. Given that each comparison was performed using quadruplicates and dye-swaps (Cy3 and Cy5 fluorochromes), our experimental design included sixteen independent competitive hybridizations (Additional file ).Transient transfections of PCCl3 cells were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), both for scrambled and for Pax8 siRNA conditions (10 ng siRNA /ml) (Dharmacon, Denver, USA). Pax8 silencing was tested by means of western blotting using a polyclonal Pax8 mouse antibody (Biopat, Milan, Italy) at different time points (24 and 48 hours) after transfection (Additional file ). Once the 48 hours condition was defined as the best time point for Pax8 silencing, we performed additional transfections to isolate total RNA using TRIzol reagent (Invitrogen, Carlsbad, CA) for each condition considered (siPax8, scrambled siRNA and PCCl3 cells treated with lipofectamine) following the manufacturer’s recommended protocol. RNA quality was evaluated with the Agilent 2100 Bioanalyzer and later amplified and labelled by using the Low RNA Input Linear Amplification Kit PLUS, Two-Color (Agilent Technologies, Palo Alto, CA). Briefly, for each sample 2 μg of total input RNA were amplified in two rounds of amplification following the manufacturer’s instructions. First strand cDNA synthesis and amplification reactions were carried out using random and T7 primers, respectively. During the 2-hour in vitro transcription, Cy3- or Cy5-labeled CTP was incorporated into each amplified RNA (cRNA). Products of the reaction were then purified using RNAeasy mini spin columns (Qiagen, Dusseldorf, Germany). Hybridization and slide and image processing were carried out according to the manufacturer’s instructions (Two-Color Microarray-Based Gene Expression Analysis protocol). In each experiment, 825 ng of contrasting cRNA samples were fragmented at 60°C for 30 min and hybridized at 65°C for 17 hr. The slides were scanned at a 10 μm resolution using the Agilent G2565BA Microarray Scanner (Agilent Technologies, Palo Alto, CA). Signal quantification was carried out with Feature Extraction 9.1 software (Agilent Technologies, Palo Alto, CA), using default analysis parameters for Agilent’s whole rat genome 44 K gene expression arrays. Array data were normalized using loess and quantile methods for normalization within and between arrays, respectively. Differential expression analysis was done using Bioconductor’s limma package. At a later stage, we used the annotate package and the data base rgug4131a.db to obtain the annotations of the rat genome from Agilent. Genes that showed adjusted p-values <0.005 were considered differentially expressed both in wild type vs. siPax8 cells and in siScramble PCCl3 vs. siPax8 PCCl3 cells. 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) under accession number GSE26938. […]

Pipeline specifications

Software tools limma, Babelomics
Application Gene expression microarray analysis
Chemicals Sodium Iodide