Kriegel Alison J, Liang Mingyu
Department of Physiology, Medical College of Wisconsin.
J Vis Exp. 2013 Nov 30(81):50785. doi: 10.3791/50785.
In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes(1), has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.
在本文中，我们描述了一种通过与地高辛标记的微小RNA探针进行原位杂交来比色检测肾脏中微小RNA的方法。该方案最初由克洛斯特曼及其同事开发，可广泛用于Exiqon微小RNA探针(1)，现已进行了修改，以克服肾脏组织中微小RNA分析所固有的挑战。这些挑战包括结构识别以及难以去除残留探针和抗体等问题。使用相对较薄的5毫米厚组织切片能够清晰观察肾脏结构，同时细胞中保留了较强的探针信号。此外，优化了探针浓度和孵育条件，以便在低背景和非特异性信号下促进微小RNA表达的可视化。在此，我们描述了优化后的方案，涵盖了从最初的组织采集和制备到程序结束时玻片封片的整个过程。该方案的基本组成部分可根据需要进行调整，以应用于其他组织和细胞培养模型。
