Abstract：
Fluorescence in situ hybridization (FISH) is a cytogenetic assay that is widely used in both clinical and research settings to validate genetic aberrations. Simple in principle, it is based on denaturation and hybridization of a DNA probe and its complementary sequence; however, it is subject to continuous optimization. Here we share how in-house FISH can be optimized using different control tissues to visualize and ultimately validate common and novel genetic abnormalities unearthed by next-generation sequencing (NGS). Seven specific FISH probes were designed and labeled, and conditions for eight tissue types and one patient-derived tumor organoid were optimized. Formalin-fixed paraffin-embedded (FFPE) tissue slides were used for each experiment. Slides were first deparaffinized, then placed in a pretreatment solution followed by a digestion step. In-house FISH probes were then added to the tissue to be denatured and hybridized, and then washed twice. To obtain optimal results, probe concentration, pepsin incubation time, denaturation, and the two post-hybridization washes were optimized for each sample. By modifying the above conditions, all FISH experiments were optimized in separate tissue types to investigate specific genomic alterations in tumors arising in those tissues. Signals were clear and distinct, allowing for visualization of the selected probes. Following this protocol, our lab has quickly optimized 11 directly labeled in-house FISH probes to support genetic aberrations nominated by NGS, including most recent discoveries through whole-genome sequencing analyses. We describe a robust approach of how to advance in-house labeled FISH probes. By following these guidelines, reliable and reproducible FISH results can be obtained to interrogate FFPE slides from benign, tumor tissues, and patient-derived tumor organoid specimens. This is of most relevance in the era of NGS and precision oncology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Metaphase FISH optimization Support Protocol 1: In-house probe labeling and preparation Support Protocol 2: Metaphase spread preparation Basic Protocol 2: Optimization of FISH on formalin-fixed paraffin-embedded tissue.
摘要：
荧光原位杂交（FISH）是一种细胞遗传学测定，广泛用于临床和研究环境中以验证遗传畸变。原则上很简单，它基于DNA探针及其互补序列的变性和杂交；然而，它受到不断优化。在这里，我们分享如何使用不同的对照组织来优化内部FISH，以可视化并最终验证下一代测序（NGS）发现的常见和新颖的遗传异常。设计并标记了7种特异性FISH探针，优化了8种组织类型和1种患者来源的肿瘤类器官的条件。每个实验使用福尔马林固定的石蜡包埋(FFPE)组织载玻片。幻灯片首先被脱石蜡，然后放入预处理溶液中，然后进行消化步骤。然后将内部FISH探针添加到要变性并杂交的组织中，然后洗了两次。为了获得最佳结果，探针浓度,胃蛋白酶孵育时间，变性,并且针对每个样品优化两次杂交后洗涤。通过修改上述条件，所有FISH实验均在不同的组织类型中进行了优化,以研究这些组织中出现的肿瘤的特定基因组改变.信号清晰清晰，允许所选探针的可视化。按照这个协议,我们的实验室快速优化了11个直接标记的内部FISH探针，以支持NGS提名的遗传畸变，包括通过全基因组测序分析的最新发现。我们描述了如何推进内部标记的FISH探针的稳健方法。通过遵循这些准则，可以获得可靠且可重复的FISH结果，以从良性，肿瘤组织，和患者来源的肿瘤类器官标本。这在NGS和精确肿瘤学时代最为相关。©2024Wiley期刊有限责任公司。基本方案1:中期FISH优化支持方案1:内部探针标记和制备支持方案2:中期铺展制备基本方案2:在福尔马林固定的石蜡包埋组织上优化FISH。
