Rehm E Jay, Hannibal Roberta L, Chaw R Crystal, Vargas-Vila Mario A, Patel Nipam H
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3140, USA.
Cold Spring Harb Protoc. 2009 Jan;2009(1):pdb.prot5130. doi: 10.1101/pdb.prot5130.
The great diversity of arthropod body plans, together with our detailed understanding of fruit fly development, makes arthropods a premier taxon for examining the evolutionary diversification of developmental patterns and hence the diversity of extant life. Crustaceans, in particular, show a remarkable range of morphologies and provide a useful outgroup to the insects. The amphipod crustacean Parhyale hawaiensis is becoming established as a model organism for developmental studies within the arthropods. This protocol describes in situ hybridization of fluorescein- or digoxigenin (DIG)-labeled RNA probes to fixed P. hawaiensis embryos. Standard techniques of molecular biology should be used to produce an appropriate template for generation of antisense RNA probes. RNA-labeling mixes designed to produce fluorescein- or DIG-labeled RNA probes using T3, T7, or SP6 RNA polymerases are commercially available. Probes should be purified using QIAGEN RNeasy columns or similar means. Considerations for double-labeling experiments using both fluorescein- and DIG-labeled RNA probes are included.
节肢动物身体结构的巨大多样性，加上我们对果蝇发育的详细了解，使节肢动物成为研究发育模式进化多样化以及现存生命多样性的首要分类群。特别是甲壳类动物，展现出了显著的形态范围，并且为昆虫提供了一个有用的外类群。夏威夷异足猛水蚤这种双足甲壳类动物正逐渐成为节肢动物发育研究的模式生物。本方案描述了用荧光素或地高辛（DIG）标记的RNA探针与固定的夏威夷异足猛水蚤胚胎进行原位杂交。应使用标准分子生物学技术来制备用于生成反义RNA探针的合适模板。设计用于使用T3、T7或SP6 RNA聚合酶生成荧光素或DIG标记RNA探针的RNA标记混合物在市面上有售。探针应使用QIAGEN RNeasy柱或类似方法进行纯化。其中还包括了使用荧光素和DIG标记的RNA探针进行双重标记实验的注意事项。
