Automated brightfield break-apart in situ hybridization (ba-ISH) application: ALK and MALT1 genes as models
Automated brightfield break-apart in situ hybridization (ba-ISH) application: ALK and MALT1 genes as models
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DOI：
10.1016/j.ymeth.2010.07.005
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发表时间：
2010-12-01
期刊：
影响因子：
4.8
通讯作者：
Grogan, Thomas M.
中科院分区：
文献类型：
--
作者：
Nitta, Hiroaki;Zhang, Wenjun;Grogan, Thomas M.
癌症诊断可能是一个复杂的过程，需要考虑组织病理学、临床、免疫表型和遗传特征。由于非随机染色体易位特别涉及各种癌症的发展，因此这些基因畸变的检测变得越来越重要。近年来，分离（或分离信号）荧光原位杂交（FISH）已成为检测组织切片上基因易位的有利技术。然而，FISH 检测在技术上具有挑战性，需要专门的荧光显微镜。此外，由于光漂白，FISH 信号对于长期存档来说并不稳定。我们的目的是证明以间变性淋巴瘤激酶 (ALK) 和粘膜相关淋巴组织易位蛋白 1 (MALT1) 基因为模型的明场分离原位杂交 (ba-ISH) 的可行性。 ALK 或 MALT1 断裂探针在已知基因断点区域的两侧用地高辛 (DIG) 或 2,4-二硝基苯基 (DNP) 标记，并结合基于碱性磷酸酶 (AP) 的蓝色和红色检测来可视化杂交位点。因此，正常基因通过混合蓝色和红色而被检测为紫色点，而易位基因被检测为孤立的蓝色或红色点。福尔马林固定、石蜡包埋的扁桃体用作共定位 5' 和 3' 探针的对照。在 ALCL 和 MALT 淋巴瘤病例中，检测到 ALK 或 MALT 基因易位为单独的蓝点和红点。因此，可以使用常规光学显微镜进行基因易位的 ISH 分析，并且可以实现分离的 ISH 载玻片的长期存档。 (C) 2010 Elsevier Inc. 保留所有权利。
Cancer diagnosis can be a complex process, which takes consideration of histopathological, clinical, immunophenotypic, and genetic features. Since non-random chromosomal translocations are specifically involved in the development of various cancers, the detection of these gene aberrations becomes increasingly important. In recent years, break-apart (or split-signal) fluorescence in situ hybridization (FISH) has emerged as an advantageous technique to detect gene translocations on tissue sections. However, FISH assays are technically challenging and require specialized fluorescence microscopes. Furthermore, the FISH signal is not stable for long term archiving due to photo bleaching. Our objective was to demonstrate the feasibility of brightfield break-apart in situ hybridization (ba-ISH) for anaplastic lymphoma kinase (ALK) and mucosa-associated lymphoid tissue translocation protein 1 (MALT1) genes as models. ALK or MALT1 break-apart probes were labeled with digoxigenin (DIG) or 2,4-dinitrophenyl (DNP) on both sides of a known gene breakpoint region and the hybridization sites were visualized with the combination of alkaline phosphatase (AP)-based blue and red detection. Therefore, normal genes are detected as purple dots by mixing blue and red colors while translocated genes are detected as isolated blue or red dots. Formalin-fixed, paraffin-embedded tonsil was used as control for the co-localized 5' and 3' probes. Gene translocations of ALK or MALT] were detected as separate blue and red dots on ALCL and MALT lymphoma cases. Thus, ISH analyses of gene translocations can be conducted with a regular light microscope and the long term archiving of break-apart ISH slides can be achieved. (C) 2010 Elsevier Inc. All rights reserved.