GSE113676
Dose-dependent transcriptomic responses of zebrafish embryos to Bisphenol A
Purpose: the goal of this project is to study the effects of the bisphenol A (BPA) in the transcriptome profiling (RNA-seq) of exposed zebrafish larvae. Methods: Total RNA was isolated from the samples using AllPrep DNA/RNA Mini Kit (Qiagen, CA, USA) as described by the manufacturer. Three high quality sample per condition were chosen to the mRNA enrichment using KAPA Stranded mRNA-Seq Kit Illumina® Platforms (Kapa Biosystems). Transcriptomic profiles were generated by deep sequencing using Illumina TruSeq SBS Kit v3-HS (pair-ended; 2x76bp) on a HiSeq2000 sequencing system. Images analysis, base calling and quality scoring of the run were processed using the manufacturer's software Real Time Analysis (RTA 1.13.48) and followed by generation of FASTQ sequence files by CASAVA. Statistical analysis: RNA-seq reads were aligned to the D. rerio reference genome (GRCz10) using STAR version 2.5.1b . Genes annotated in GRCz10.84 were quantified using RSEM version 1.2.28 with default parameters. Differential expression analysis between all BPA conditions was performed with the DESeq2 (v.1.10.1) R package with the Likelihood ratio test option. ANOVA-PLS was performed on the normalized data using the lmdme package in R (v. 1.0.136, R Core Team). Results: We generated on average 40 million paired-end reads for each sample and identified aproximatelly 25000 transcripts. 2539 differentially expressed genes (DEGs) were detected which could be divided in 3 clusters including 960, 1132 and 447 genes, respectively. Affected metabolic pathways were analyzed from the DEGs: liver development, lipid transport, lysosome and protein glycosylation, metabolic pathways for lipids, glutathione, retinol, and steroid hormones, and cytochrome P450-mediated metabolism in adittion to protein ubiquitination and glycolysis/gluconeogenesis pathways' were down or upregulated. Conclusions: The results suggest the interaction of BPA with different signaling pathways, being the estrogenic and retinoid receptors two likely BPA targets.  We concluded that our analysis allowed us the identification of underlying molecular mechanisms occurring simultaneously at the exposed animals. While this approach is very useful to analyze the effects of compounds, like BPA, able to interact with different cellular targets, we believe that it can be also applied to the characterization of the different toxic components present in complex natural mixtures. Overall design: Whole embryo (5 dpf; wild type zebrafish) mRNA profiles of 4 groups (control, 0.1, 1 and 4 ppm of BPA) were generated by deep sequencing, in triplicate, using Illumina TruSeq SBS Kit v3-HS (pair-ended) on a HiSeq2000 sequencing system.