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2017 ; 8
(31
): 50534-50541
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A molecular pathology method for sequential fluorescence in situ hybridization
for multi-gene analysis at the single-cell level
#MMPMID28881581
Hu L
; Yin X
; Sun J
; Zetterberg A
; Miao W
; Cheng T
Oncotarget
2017[Aug]; 8
(31
): 50534-50541
PMID28881581
show ga
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.