全山RNA荧光<em在原位</em>杂交<em>果蝇</em>胚胎
Summary
在这里，我们描述了一个全型日光灯<em在原位</em杂交（FISH）协议确定的RNA表达在果蝇胚胎发育过程中的表达和定位性能，<em>果蝇</em>。
Abstract
评估的表达模式的基因，以及其转录RNA的亚细胞定位性能，了解其生物学功能在开发过程中的关键功能。 RNA 原位杂交（RNA-ISH）是一个功能强大的方法，用于可视化的RNA分布特性，无论是在有机体，细胞或亚细胞水平的1。 RNA-ISH基于杂交的标记的核酸探针（ 例如，反义RNA，寡核苷酸）互补的mRNA的序列，或一个非编码RNA靶景点2。由于该过程要求单独初级序列信息来生成序列特异性探针，它可以普遍适用于范围广泛的生物体和组织标本3。事实上，一些大型ISH研究已实施的记录在各种模式生物的基因表达和RNA的本地化动态，这导致了重要的社会资源，建立4-11。虽然多年来，已开发各种探针标记和检测策略的结合使用荧光标记的检测试剂和酶的信号放大步骤提供显着增强的灵敏度和分辨率的程序12。在这里，我们描述了一个优化的荧光原位杂交法（FISH）采用酪胺信号放大（TSA）中上演了果蝇胚胎的RNA表达和定位动态可视化。该过程进行96孔PCR板格式，极大地方便了同时处理大量的样品。
Protocol
1。 RNA探针制备 概述：以下部分介绍了所需要的步骤，使地高辛（DIG）标记的RNA探针适合鱼类。第一步涉及克隆或PCR扩增的序列对应于感兴趣的基因的转录区域，将用于生成一个序列特异性探针。这可以通过以下来实现第一个克隆的基因片段插入的质粒，其中的多个克隆位点的两侧是噬菌体启动子元件（T7，T3或SP6），然后可以使用侧翼结合的启动子序列的引物，?…
Representative Results
当成功执行，这个程序提供了一个在果蝇胚胎发育早期的基因表达和mRNA的定位动态的时空分析的详细程度显着增强。事实上， 如图3A所示为经典的配对规则基因欠幅脉冲 （ 运行 ），可以使用这个协议来观察基因表达事件通过检测组新生成绩单灶的表达核。此外，如胚胎镶嵌在图3B中所示，该方法能够在高分辨率的mRNA的定位功能的可视化。 <p class=…
Discussion
探针的合成步骤，我们通常会产生径流的反义RNA探针， 通过体外转录全长果蝇的cDNA扩增出质粒发现在果蝇的基因收藏（DGC），资源详载于以下网址： http://www .fruitfly.org / DGC / index.html的 14,15的 。这种方法已被广泛使用在的大型原位杂交研究，目的是在果蝇胚胎9,16测绘基因的表达和mRNA的定位模式。 DGC的质粒?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
由国家科学和工程委员会（NSERC），加拿大，加拿大卫生研究院（CIHR）和德Recherche全宗连接桑特杜魁北克省（FRSQ）的资助下进行的工作在Lécuyer实验室的支持。法比奥·亚历克西斯列斐伏尔和盖尔Moquin丽怡NSERC本科生科研奖学金的支持，，而卡罗尔Iampietro所支持的的安杰洛Pizzagalli的博士后奖学金。
Materials
| Name of the Reagent | Company | Catalogue Number | Comments (optional) |
| T7, T3 or SP6 RNA Polymerase | Fermentas Life Sciences | EP0101,EP0111,EP0131 | Kits contain reaction buffer. |
| DIG RNA Labeling Mix | Roche Applied Science | 11 277 073 910 | |
| RNAguard | Amersham Biosciences | 27-0816-01 | |
| 3M sodium acetate | |||
| Cold 100% ethanol. | |||
| Cold 70% ethanol. | |||
| Chlorine bleach solution diluted 1:1 with water. | |||
| Heptane | |||
| Methanol | |||
| proteinase K | Sigma Aldrich Oakville, ON, Canada | Catalog No. P2308 | |
| 40% formaldehyde solution, freshly prepared | |||
| PBS-Tween solution (PBT) | 1xPBS, 0.1% Tween-20 | ||
| Glycine solution | 2 mg/ ml glycine in PBT | ||
| HRP-conjugated mouse monoclonal anti-DIG | Jackson ImmunoResearch Laboratories Inc | 200-032- 156 | (1/400 dilution of a 1 mg/ml stock solution in PBTB |
| HRP-conjugated sheep monoclonal anti-DIG | Roche Applied Science, Laval, QC | 1 207 733 | 1/500 dilution of stock solution in PBTB |
| Biotin-conjugated mouse monoclonal anti-DIG | Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA | 200-062-156 | (1/400 dilution of a 1 mg/ml stock solution in PBTB |
| Streptavidin-HRP conjugate | Molecular Probes, Eugene OR, USA | S991 | (1/100 dilution of a 1 μg/ml stock |
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Citazione di questo articolo
Legendre, F., Cody, N., Iampietro, C., Bergalet, J., Lefebvre, F. A., Moquin-Beaudry, G., Zhang, O., Wang, X., Lécuyer, E. Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos. J. Vis. Exp. (71), e50057, doi:10.3791/50057 (2013).