Research Papers:

A molecular pathology method for sequential fluorescence in situ hybridization for multi-gene analysis at the single-cell level

Linping Hu, Xiuxiu Yin, Jiangman Sun, Anders Zetterberg, Weimin Miao and Tao Cheng _

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Oncotarget. 2017; 8:50534-50541. https://doi.org/10.18632/oncotarget.10245

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Linping Hu1, Xiuxiu Yin1, Jiangman Sun1, Anders Zetterberg2, Weimin Miao1,3 and Tao Cheng1

1State Key Laboratory of Experimental Hematology, Institute of Hematology, Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin China

2The Department of Oncology-Pathology, Karolinska Cancer Institute, Karolinska Institute, Stockholm, Sweden

3Union Stem Cell and Gene Engineering Co. Ltd, Tianjin China

Correspondence to:

Tao Cheng, email: [email protected]

Weimin Miao, email: [email protected]

Keywords: molecular pathology, genotyping, sequential FISH, single cell, multi-gene detection

Received: November 10, 2015     Accepted: May 01, 2016     Published: June 23, 2016


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.

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