A molecular pathology method for sequential fluorescence in situ hybridization for multi-gene analysis at the single-cell level
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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
Tao Cheng, email: email@example.com
Weimin Miao, email: firstname.lastname@example.org
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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