A molecular pathology method for sequential fluorescence in situ hybridization for multi-gene analysis at the single-cell level.
A molecular pathology method for sequential fluorescence in situ hybridization for multi-gene analysis at the single-cell level.
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一种用于单细胞水平多基因分析的连续荧光原位杂交的分子病理学方法。
DOI：
10.18632/oncotarget.10245
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发表时间：
2017-08-01
期刊：
影响因子：
--
通讯作者：
Cheng T
中科院分区：
文献类型：
--
作者：
Hu L;Yin X;Sun J;Zetterberg A;Miao W;Cheng T
单细胞水平的多基因检测是期望的，以使得能够对异质血液学和肿瘤学样品进行更精确的基因分型。本研究旨在建立一种单细胞多基因荧光原位杂交（FISH）方法，用于分子病理学分析。用5种荧光染料标记不同的FISH基因探针，用5种颜色的FISH方案检测5个基因。第一次杂交后，剥离先前的FISH探针组，并使用第二组五色FISH探针进行再杂交。每次杂交后，在6个荧光过滤器通道中记录荧光信号，所述通道包括DAPI、Spectrum绿色™、Cy 3 ™ v1、德克萨斯红、Cy 5和PF-415。使用数字自动重新定位程序以确保在每个剥离和杂交循环中研究完全相同的显微镜视野。通过使用这种顺序剥离和再杂交策略，可以在单个细胞核内检测到多达20个基因。总之，一个实用的分子病理学方法，在单细胞水平上分析多个基因。
Multi-gene detection at the single-cell level is desirable to enable more precise genotyping of heterogeneous hematology and oncology samples. This study aimed to establish a single-cell multi-gene fluorescence in situ hybridization (FISH) method for use in molecular pathology analyses. Five fluorochromes were used to label different FISH gene probes, and 5 genes were detected using a five-color FISH protocol. After the first hybridization, the previous FISH probe set was stripped, and a second set of five-color FISH probes was used for rehybridization. After each hybridization, the fluorescence signals were recorded in 6 fluorescence filter channels that included DAPI, Spectrum Green™, Cy3™ v1, Texas Red, Cy5, and PF-415. A digital automatic relocation procedure was used to ensure that exactly the same microscopic field was studied in each stripping and hybridization cycle. By using this sequential stripping and rehybridization strategy, up to 20 genes can be detected within a single nucleus. In conclusion, a practical molecular pathology method was developed for analyzing multiple genes at the single-cell level.