A method for conducting highly sensitive microRNA in situ hybridization and immunohistochemical analysis in pancreatic cancer.
A method for conducting highly sensitive microRNA in situ hybridization and immunohistochemical analysis in pancreatic cancer.
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DOI：
10.1007/978-1-62703-287-2_4
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发表时间：
2013-01-01
期刊：
影响因子：
--
通讯作者：
Korc, Murray
中科院分区：
文献类型：
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作者：
Sempere, Lorenzo F;Korc, Murray
全组织活检的分析实验已将 microRNA (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，肿瘤病变的不同细胞区室（癌细胞和非癌细胞）中可能发生 miRNA 表达的改变，因此确定 PDAC 组织中哪种特定细胞类型表达特定 miRNA 非常重要。在这里，我们描述了一种高度灵敏的基于荧光的 ISH 方法，使用 5' 和 3' 末端荧光素标记的锁定核酸 (LNA) 修饰探针，在福尔马林固定石蜡包埋 (FFPE) 组织切片中可视化单个细胞内 miRNA 的积累。我们描述了一种多色 ISH/IHC 方法，该方法基于辣根过氧化物酶 (HRP) 介导的酪酰胺信号放大 (TSA) 反应，使用不同的内部合成的荧光染料缀合底物，能够共同检测 miRNA、丰富的非编码 RNA 和蛋白质标记，用于信号定量以及 FFPE 中的细胞类型共定位研究 来自临床标本和 PDAC 小鼠模型的胰腺组织切片。
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.