Department of Pathology and Laboratory Medicine, Division of Neuropathology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
Nat Protoc. 2010 Jun;5(6):1061-73. doi: 10.1038/nprot.2010.62. Epub 2010 May 20.
Noncoding RNAs (ncRNAs) comprise a diverse group of RNAs that function in essential cellular processes such as pre-mRNA splicing and mRNA translation and also regulate various aspects of gene expression in physiology and development. Methods of subcellular and tissue localization of ncRNAs are essential to understand their biological roles and their contribution to disease. We describe a rapid fluorescent (FISH) or chromogenic (CISH) in situ hybridization protocol for localization of ncRNAs (including microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), piwi-associated RNAs (piRNAs) and ribosomal RNAs (rRNAs)) in formalin-fixed, paraffin-embedded (FFPE) tissues and cultured cells, using locked nucleic acid (LNA)-modified oligonucleotides. In this protocol, sections are heated in citrate buffer, which eliminates the need for protease treatment, thus preserving optimal morphology and protein epitopes, and allowing the simultaneous detection of proteins with immunofluorescence staining (IF). LNA-FISH requires 5 h, or between 10 and 36 h when combined with IF; LNA-CISH requires 2 d.
非编码 RNA（ncRNAs）是一组功能多样的 RNA，它们在包括前体 mRNA 剪接和 mRNA 翻译在内的重要细胞过程中发挥作用，也调节生理和发育过程中基因表达的各个方面。ncRNAs 的亚细胞和组织定位方法对于理解它们的生物学功能及其对疾病的贡献至关重要。我们描述了一种快速荧光原位杂交（FISH）或显色原位杂交（CISH）方法，用于定位福尔马林固定、石蜡包埋（FFPE）组织和培养细胞中的 ncRNAs（包括 microRNAs（miRNAs）、小核仁 RNA（snoRNAs）、小核 RNA（snRNAs）、piwi 相关 RNA（piRNAs）和核糖体 RNA（rRNAs）），使用锁核酸（LNA）修饰的寡核苷酸。在该方案中，切片在柠檬酸缓冲液中加热，这消除了蛋白酶处理的需要，从而保持了最佳的形态和蛋白质表位，并允许与免疫荧光染色（IF）同时检测蛋白质。LNA-FISH 需要 5 小时，与 IF 结合时需要 10 到 36 小时；LNA-CISH 需要 2 天。
