Rapid in situ codetection of noncoding RNAs and proteins in cells and formalin-fixed paraffin-embedded tissue sections without protease treatment
Rapid in situ codetection of noncoding RNAs and proteins in cells and formalin-fixed paraffin-embedded tissue sections without protease treatment
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DOI：
10.1038/nprot.2010.62
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发表时间：
2010-01-01
期刊：
影响因子：
14.8
通讯作者：
Mourelatos, Zissimos
中科院分区：
文献类型：
--
作者：
de Planell-Saguer, Mariangels;Celina Rodicio, Maria;Mourelatos, Zissimos
非编码RNA（NCRNA）组成了一组不同的RNA，它们在基本细胞过程中起作用，例如MRNA剪接和mRNA翻译，还调节了生理学和发育中基因表达的各个方面。 NCRNA的亚细胞和组织定位方法对于了解其生物学作用及其对疾病的贡献至关重要。 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在福尔马林固定，石蜡包裹的核糖体RNA（rRNA）） （FFPE）组织和培养细胞，使用锁定的核酸（LNA）修饰的寡核苷酸。在该方案中，切片在柠檬酸盐缓冲液中加热，这消除了对蛋白酶处理的需求，从而保留最佳形态和蛋白质表位，并允许同时检测具有免疫荧光染色的蛋白质（如果）。与if结合时，LNA-FISH需要5小时或在10到36小时之间； LNA-cish需要2 d。
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.