Wang Rong-Lin, Biales Adam, Bencic David, Lattier David, Kostich Mitch, Villeneuve Dan, Ankley Gerald T, Lazorchak Jim, Toth Greg
Ecological Exposure Research Division, National Exposure Research Laboratory, US Environmental Protection Agency, Cincinnati, Ohio 45268, USA.
Environ Toxicol Chem. 2008 Mar;27(3):652-63. doi: 10.1897/07-191.1.
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.
本文所呈现的研究是一项关于内分泌干扰化学物质对斑马鱼（Danio rerio）下丘脑 - 垂体 - 性腺轴分子作用模式的更大规模研究的一部分。它探讨了对微阵列在水生生态毒理学应用至关重要的几个问题：实验设计、微阵列扫描、基因表达强度分布以及实验参数对斑马鱼转录组的影响。使用安捷伦寡核苷酸微阵列（G2518A）检测了暴露于17α - 乙炔雌二醇（30 ng/L）、法倔唑（25 μg/L）或17β - 群勃龙（3.0 μg/L）48小时或96小时的单个斑马鱼不同组织的表达谱。作为微阵列研究中常用设计的一种灵活且高效的替代方案，发现不平衡不完全区组设计非常适合这项工作，高数据重现性、低微阵列间变异性以及几乎没有基因特异性染料偏差都证明了这一点。随机扫描仪噪声对数据重现性影响很小。通过高光谱成像揭示了低水平、略有变化的花青素3（Cy3）污染物，这表明荧光污染可能是与弱表达基因相关的大变异的一个潜在因素。斑马鱼基因的表达强度偏向于其分布范围的下限，并且更多弱表达基因往往具有更大的变异。组织类型对总体基因表达谱影响最大，其次是性别、化学处理和暴露持续时间，这一发现对于实验设计优化可能至关重要。总体而言，在20对卵巢样本的一个子集中检测的13个基因的定量聚合酶链反应结果与微阵列谱之间的一致性非常好。这些发现将有助于改进微阵列在未来生态毒理学研究中的应用。
