Sempere Lorenzo F, Korc Murray
Department of Medicine, Dartmouth Hitchcock Medical Center, Hanover, NH, USA.
Methods Mol Biol. 2013;980:43-59. doi: 10.1007/978-1-62703-287-2_4.
Profiling experiments in whole tissue biopsies have linked altered expression of microRNAs (miRNAs) to different types of cancer, including pancreatic ductal adenocarcinoma (PDAC). Emerging evidence indicates that altered miRNA expression can occur in different cellular compartments (cancer and non-cancer cells) in tumor lesions, and thus it is important to ascertain which specific cell type expresses a particulars miRNA in PDAC tissues. Here, we describe a highly sensitive fluorescence-based ISH method to visualize miRNA accumulation within individual cells in formalin-fixed paraffin-embedded (FFPE) tissue sections using 5' and 3' terminally fluorescein-labeled locked nucleic acid (LNA)-modified probes. We describe a multicolor ISH/IHC method based on sequential rounds of horseradish peroxidase (HRP)-mediated tyramide signal amplification (TSA) reactions with different in-house synthesized fluorochrome-conjugated substrates that enable co-detection of miRNAs, abundant noncoding RNAs and protein markers for signal quantification, and cell type co-localization studies in FFPE pancreatic tissue sections from clinical specimens and mouse models of PDAC.
全组织活检中的分析实验已将微小RNA（miRNA）表达的改变与不同类型的癌症联系起来，包括胰腺导管腺癌（PDAC）。新出现的证据表明，miRNA表达的改变可发生在肿瘤病变的不同细胞区室（癌细胞和非癌细胞）中，因此确定PDAC组织中哪种特定细胞类型表达特定的miRNA很重要。在这里，我们描述了一种基于荧光的高灵敏度原位杂交方法，使用5'和3'末端荧光素标记的锁核酸（LNA）修饰探针，在福尔马林固定石蜡包埋（FFPE）组织切片的单个细胞内可视化miRNA积累。我们描述了一种基于多轮辣根过氧化物酶（HRP）介导的酪胺信号放大（TSA）反应的多色原位杂交/免疫组化方法，该反应使用不同的内部合成荧光染料偶联底物，能够在来自临床标本和PDAC小鼠模型的FFPE胰腺组织切片中共同检测miRNA、丰富的非编码RNA和蛋白质标记物，用于信号定量以及细胞类型共定位研究。
