DNA microarray application in ecotoxicology: Experimental design, microarray scanning, and factors affecting transcriptional profiles in a small fish species

Abstract

The research presented here is part of a larger study of the molecular mode of action of endocrine-disrupting chemicals targeting the hypothalamic-pituitary-gonadal axis in zebrafish (Danio rerio). It addresses several issues critical to microarray application in aquatic ecotoxicology: experimental design, microarray scanning, gene expression intensity distribution, and the effect of experimental parameters on the zebrafish transcriptome. Expression profiles from various tissues of individual zebrafish exposed to 17alpha-ethinylestradiol (30 ng/L), fadrozole (25 micro.g/L), or 17beta-trenbolone (3.0 microg/L) for 48 or 96 h were examined with the Agilent Oligo Microarray (G2518A). As a flexible and efficient alternative to the designs commonly used in microarray studies, an unbalanced incomplete block design was found to be well suited for this work, as evidenced by high data reproducibility, low microarray-to-microarray variability, and little gene-specific dye bias. Random scanner noise had little effect on data reproducibility. A low-level, slightly variable Cyanine 3 (Cy3) contaminant was revealed by hyperspectral imaging, suggesting fluorescence contamination as a potential contributor to the large variance associated with weakly expressed genes. Expression intensities of zebrafish genes were skewed toward the lower end of their distribution range, and more weakly expressed genes tended to have larger variances. Tissue type, followed in descending order by gender, chemical treatment, and exposure duration, had the greatest effect on the overall gene expression profiles, a finding potentially critical to experimental design optimization. Overall, congruence was excellent between quantitative polymerase chain reaction results and microarray profiles of 13 genes examined across a subset of 20 pairs of ovarian samples. These findings will help to improve applications of microarrays in future ecotoxicological studies.

Implications of the solvent vehicles dimethylformamide and dimethylsulfoxide for establishing transcriptomic endpoints in the zebrafish embryo toxicity test

“…Gene expression profiling techniques are actively being assessed for use in alternative regulatory test procedures such as the zebrafish embryo test [17,18]. However, acceptance will require a thorough understanding of confounding factors and artefacts, including potential effects of solvents and solvent-toxicant interactions, so that true toxicant effects can be separated from baseline characteristics [19].…”

Applications of biological tools or biomarkers in aquatic biota: A case study of the Tamar estuary, South West England

“…Consequently, biological tools of increasing utility in ERA are microarrays, which have been developed for several of the species commonly used as sentinels within the Tamar, including M. edulis (Canesi et al, 2011;Domeneghetti et al, 2011;Dondero et al, 2006;Place et al, 2008;, C. gigas (Dheilly et al, 2011), N. virens (Last and Rosato, 2004) and C. maenas (Towle et al, 2011). Such arrays consist of many oligonucleotide probes attached to a solid surface or 'chip' and enable simultaneous measurement of expression for multiple genes (Neumann and Galvez, 2002;Wang et al, 2008). This approach is particularly useful where little is known about the mechanisms of action or predicted responses to a given contaminant.…”

“…2). It is, however, especially important to have robust controls and baseline data when using such sensitive tools (Neumann and Galvez, 2002;Wang et al, 2008). No other microarray studies have been performed in the Tamar as yet, but their use in relevant species is increasing.…”

Dietary sodium protects fish against copper-induced olfactory impairment

“…The other reason could be that they pooled the olfactory rosettes, olfactory nerves, telecephalon and the olfactory bulbs whereas in our study we used just olfactory rosettes. Changes at transcriptional level could be different at different tissues (Wang et al, 2008). In addition, in the current study we used yellow perch, a metal tolerant fish (Taylor et al, 2003;Couture and Pyle, 2008).…”