Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification
Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification
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DOI：
10.1038/nprot.2007.313
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Kauppinen, Sakari
中科院分区：
文献类型：
--
作者：
Silahtaroglu, Asli N.;Nolting, Dorrit;Kauppinen, Sakari
在组织、细胞和亚细胞水平上确定空间和时间microRNA（miRNA）积累的能力对于理解miRNA和miRNA相关基因调控网络的生物学作用是必不可少的。该方案描述了一种使用荧光原位杂交（FISH）快速有效地检测冷冻组织切片中miRNA的方法。该方法将锁核酸（LNA）修饰的寡核苷酸探针的独特miRNA识别特性与使用酪胺信号放大（TSA）技术的FISH相结合。虽然这两种方法以前已经被证明可以提高FISH的检测灵敏度，但将这些技术结合到一个方案中可以显著减少冷冻切片中miRNA检测所需的时间，同时保持高检测灵敏度。从组织切片的固定开始，该miRNA FISH方案可以在大约6小时内完成，并且允许以高细胞分辨率在各种动物组织冷冻切片以及人类肿瘤活检中检测miRNA。
The ability to determine spatial and temporal microRNA (miRNA) accumulation at the tissue, cell and subcellular levels is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. This protocol describes a method for fast and effective detection of miRNAs in frozen tissue sections using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA)-modified oligonucleotide probes with FISH using the tyramide signal amplification (TSA) technology. Although both approaches have previously been shown to increase detection sensitivity in FISH, combining these techniques into one protocol significantly decreases the time needed for miRNA detection in cryosections, while simultaneously retaining high detection sensitivity. Starting with fixation of the tissue sections, this miRNA FISH protocol can be completed within approximately 6 h and allows miRNA detection in a wide variety of animal tissue cryosections as well as in human tumor biopsies at high cellular resolution.