Lan Chuan-Ching, Tang Rongying, Un San Leong Ivone, Love Donald R
School of Biological Sciences, The University of Auckland, Auckland 1142, New Zealand.
Cold Spring Harb Protoc. 2009 Oct;2009(10):pdb.prot5314. doi: 10.1101/pdb.prot5314.
The zebrafish (Danio rerio) has emerged as a popular model species. The rapid development of zebrafish embryos provides opportunities for investigation of genes essential for developmental processes, the human counterparts of which might be implicated in diseases. Understanding when and where genes are expressed can facilitate greater understanding of their function, and also allow the genes to be manipulated by gene knockdown in temporally and spatially specific manners. Quantitative real-time polymerase chain reaction (qRT-PCR) is widely applied in gene expression studies. This protocol presents techniques to optimize RNA isolation from zebrafish embryos; quality assessment and the use of multiple reference genes are also emphasized. The combined use of TRIzol extraction and column-based purification is strongly recommended, because the resulting RNA is of better quality than RNA isolated using either of those methods alone. The procedure can be performed in 2 d, with individual stages taking up to 15 h to complete.
斑马鱼（Danio rerio）已成为一种广受欢迎的模式生物。斑马鱼胚胎的快速发育为研究发育过程中必需的基因提供了机会，而这些基因在人类中的对应物可能与疾病有关。了解基因何时何地表达有助于更深入地理解其功能，还能通过时空特异性的基因敲低来操纵这些基因。定量实时聚合酶链反应（qRT-PCR）广泛应用于基因表达研究。本方案介绍了从斑马鱼胚胎中优化RNA提取的技术；同时也强调了质量评估和多个参考基因的使用。强烈建议联合使用TRIzol提取法和基于柱的纯化法，因为由此获得的RNA质量比单独使用这两种方法中的任何一种所分离的RNA质量更好。该实验可在两天内完成，各个阶段最多需要15小时。
