The Comprehensive Cancer Center, Ohio State University Medical Center, Columbus, OH, USA.
Methods. 2010 Dec;52(4):307-15. doi: 10.1016/j.ymeth.2010.08.009. Epub 2010 Aug 17.
This manuscript details a protocol for the co-localization of a microRNA and its putative protein target in paraffin embedded formalin fixed tissues. The key variables for the first step, microRNA in situ hybridization, includes probe concentration (1-2 pmol/μl), locked nucleic acid (LNA) modified probes, protease digestion (pepsin 1.3mg/ml), and a low stringency wash. Key variables for the subsequent immunohistochemical step are the concentration of the primary antibody, proper pretreatment (none, proteinase K, or antigen retrieval), and use of a highly sensitive detection system. A computer based system can convert the colorimetric signals (blue chromogen (NBT/BCIP) for the microRNA, and either a red (fast red) or brown (DAB) chromogen for the protein) to distinct fluorescent-based colors, and then mix them to determine if a given cell has the microRNA and protein of interest. Co-expression of a microRNA and its putative target in tissue sections offers physiologic corroboration of solution-based methods that a given microRNA may be regulating a specific protein.
本文详细介绍了一种在石蜡包埋福尔马林固定组织中检测 microRNA 与其假定蛋白靶标的共定位的方案。该方案中，microRNA 原位杂交的关键变量包括探针浓度（1-2 pmol/μl）、锁核酸（LNA）修饰探针、蛋白酶消化（胃蛋白酶 1.3mg/ml）和低严格性洗涤。后续免疫组织化学步骤的关键变量是一抗的浓度、适当的预处理（无预处理、蛋白酶 K 处理或抗原修复）以及使用高灵敏度的检测系统。基于计算机的系统可以将比色信号（microRNA 的蓝色显色剂（NBT/BCIP）和蛋白的红色（快速红）或棕色（DAB）显色剂）转换为不同的荧光颜色，然后混合这些颜色以确定给定细胞是否具有感兴趣的 microRNA 和蛋白。microRNA 与其假定靶标在组织切片中的共表达为基于溶液的方法提供了生理佐证，即特定的 microRNA 可能调控特定的蛋白。
