Computational protocol: The Transcriptional Response to Oxidative Stress during Vertebrate Development: Effects of tert-Butylhydroquinone and 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

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Protocol publication

[…] We examined a subset of RNA samples from Experiment 1 by microarray using the Agilent 22k long-oligo zebrafish array. We analyzed all of the 4-dpf samples: four biological replicates each for DMSO-, tBHQ- and TCDD-treated eleutheroembryos, each hybridized against a universal reference mRNA created from equal amounts of RNA from 2 replicates each from all toxicants (TCDD, tBHQ, DMSO) and time points (1, 2, 3, 4, 5, 6-dpf). The use of a universal reference RNA balances efficiency with statistical power and has several advantages –. It facilitates normalization because all of the genes expressed in experimental samples are represented in the reference samples . Dye bias is minimized because all experimental samples are labeled with the same dye; thus, dye swaps are not needed . To verify this, we performed quality control hybridizations including a dye swap and a self-self hybridization. Analysis of a self-self hybridization of the Universal Reference (composed of equal amounts of RNA from all timepoints, toxicants, and replicates) revealed 18293 of 21495 features (85%) with signal above background (calculated as 2.6 times the background standard deviation). This indicates that the majority of the probes on the Agilent microarray represent transcripts expressed in 1–6 dpf embryos.RNA samples from 4-dpf eleutheroembryos treated with DMSO, tBHQ, or TCDD were checked for quality using a NanoDrop ND-1000 spectrophotometer and an Agilent 2100 BioAnalyzer. cDNA synthesis from 200 ng of total RNA was performed using the Agilent Low RNA Input Linear Fluorescent Amplification Plus kit, following the manufacturer's instructions. cRNA was synthesized from the cDNA template, with incorporation of either cyanine-5-CTP or cyanine-3-CTP (Perkin Elmer). Labeled cRNA was purified using the Qiagen RNeasy Mini kit; quantity and quality was assessed by NanoDrop spectrophotometer and Agilent BioAnalyzer. cRNA samples were hybridized to Agilent 22k zebrafish microarrays using the Agilent Gene Expression Hybridization Kit. An aliquot (750 ng) of each cy5-labeled sample cRNA was hybridized against 750 ng of cy3-labeled cRNA derived from the Universal mRNA Reference. Labeled cRNAs were combined with the Agilent 25× fragmentation buffer and incubated at 60°C for 30 minutes. This was followed by mixing with 2× hybridization buffer, after which 100 µl of the product was spread evenly across the surface of an Agilent 22K zebrafish microarray. The loaded microarray was incubated at 60°C for 17 hours with rotation in an Agilent DNA Microarray Hybridization Oven. Post-hybridization, microarray slides were washed, air-dried, and stored in darkness with desiccation prior to laser-excited fluorescence scanning in an Agilent DNA Microarray Scanner.Analysis of raw microarray data was performed using Agilent's feature extraction protocol, which includes spot finding, spot analysis, background subtraction (using local background plus global background based on spots along the central tendency line for red versus green intensity), dye normalization (linear and lowess algorithms, using spots along the central tendency line as for background subtraction), and final calculation of Cy5/Cy3 ratios and log2 transformed fold change for each spot. Features with signal not significantly above background, non-uniform features, and features exhibiting saturation were flagged. The microarray data have been deposited in MIAME-compliant format in the Gene Expression Omnibus (GEO) database at the U.S. National Center for Biotechnology Information (accession number GSE10157; http://www.ncbi.nlm.nih.gov/geo/).Probes indicating significantly different relative transcript abundance among the DMSO, TCDD, and tBHQ treatments were determined by Analysis of Variance (ANOVA) using MEV in the TM4 suite of microarray software , . Data were first log transformed and values for each probe median centered. ANOVA was performed with a distribution based on 1000 permutations of the data, a significance value of p<0.05, and control of False Discovery Rate (FDR) at 5% , . An objective of the statistical analysis was to minimize type II error while maintaining a reasonable false discovery rate.Hierarchical cluster analysis of the significant probes was performed using CLUSTER software and log2 transformed fold change values were median centered for both probes and microarrays. Cluster analysis used Pearson's correlation (i.e. centered) and average linkage clustering. Enrichment of Gene Ontology (GO) terms for clusters of significant probes thus identified was examined using the FatiGO+ software . FatiGO+ uses GO terms assigned within the Ensembl annotation of the zebrafish genome and the background set of probes used in each analysis was the entire probe set of the Agilent microarray less the probes found significant by the ANOVA analysis described above. […]

Pipeline specifications

Software tools TM4, Babelomics
Databases GEO
Application Gene expression microarray analysis
Organisms Danio rerio
Diseases Cardiovascular Diseases, Drug-Related Side Effects and Adverse Reactions
Chemicals Dimethyl Sulfoxide, Glutathione