Salehi Reza, Tsoi Stephen C M, Colazo Marcos G, Ambrose Divakar J, Robert Claude, Dyck Michael K
Department of Agricultural, Food and Nutritional Science, University of Alberta.
Livestock Research Branch, Alberta Agriculture and Forestry.
J Vis Exp. 2017 Jan 30(119):53754. doi: 10.3791/53754.
Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.
早期胚胎损失是导致牛不育的一个重要因素。此外，由于牛的发育过程与人类相似，它成为研究人类植入前胚胎发育的一个有趣模型。尽管已知遗传因素会影响早期胚胎发育，但发现这些因素一直是一项严峻挑战。微阵列技术允许在全基因组范围内对转录水平进行定量测量和基因表达谱分析。在规划微阵列实验时必须做出的主要决策之一是使用单色还是双色方法。双色设计增加了技术重复，最小化了变异性，提高了灵敏度和准确性，还允许进行循环设计，定义共同参考样本。尽管微阵列是一种强大的生物学工具，但也存在一些可能削弱其功效的潜在陷阱。因此，在本技术论文中，我们展示了一种优化方案，该方案涉及RNA提取、扩增、标记、将标记的扩增RNA与阵列杂交、阵列扫描以及使用双色分析策略进行数据分析。
