Fluorescent In Situ Hybridization in Suspension by Imaging Flow Cytometry

Maguire, Orla; Wallace, Paul K.; Minderman, Hans

doi:10.1007/978-1-4939-3302-0_7

Cited by 12 publications

(9 citation statements)

References 11 publications

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“…We expect more advanced light-sheet microscopy setups ( 39 – 42 ) to perform equally well or better but they would require a specialized sample chamber for side-illumination. In principle, imaging flow cytometers with an extended depth of field can be used to increase throughput for counting fluorescent spots ( 43 ). Whether their sensitivity is sufficient to measure sFISH spots needs to be tested in the future.…”

Section: Discussionmentioning

confidence: 99%

mRNA detection in budding yeast with single fluorophores

Wadsworth

Parikh

Choy

et al. 2017

Nucleic Acids Research

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Quantitative measurement of mRNA levels in single cells is necessary to understand phenotypic variability within an otherwise isogenic population of cells. Single-molecule mRNA Fluorescence In Situ Hybridization (FISH) has been established as the standard method for this purpose, but current protocols require a long region of mRNA to be targeted by multiple DNA probes. Here, we introduce a new single-probe FISH protocol termed sFISH for budding yeast, Saccharomyces cerevisiae using a single DNA probe labeled with a single fluorophore. In sFISH, we markedly improved probe specificity and signal-to-background ratio by using methanol fixation and inclined laser illumination. We show that sFISH reports mRNA changes that correspond to protein levels and gene copy number. Using this new FISH protocol, we can detect >50% of the total target mRNA. We also demonstrate the versatility of sFISH using FRET detection and mRNA isoform profiling as examples. Our FISH protocol with single-fluorophore sensitivity significantly reduces cost and time compared to the conventional FISH protocols and opens up new opportunities to investigate small changes in RNA at the single cell level.

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Section: Discussionmentioning

confidence: 99%

mRNA detection in budding yeast with single fluorophores

Wadsworth

Parikh

Choy

et al. 2017

Nucleic Acids Research

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“…The following method was adapted from Minderman et al (2012) . Each BAC clone at 60 ng (giving a total of 1 µg) was combined with 50 µg Cot1 DNA (Abbott Molecular, NSW, Australia), desiccated at 45°C for 30 min, followed by resuspension in 20 µL of locus‐specific identifier (LSI) hybridization buffer (Abbott Molecular, NSW, Australia).…”

Section: Methodsmentioning

confidence: 99%

Development of locus specific sub‐clone separation by fluorescence in situ hybridization in suspension in chronic lymphocytic leukemia

Bailey

Macardle

et al. 2017

Cytometry Pt A

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Intra-tumor genetic heterogeneity is a hallmark of cancer. The ability to monitor and analyze these sub-clonal cell populations can be considered key to successful treatment, particularly in the modern era of targeted therapies. Although advances in sequencing technologies have significantly improved our ability to analyze the mutational landscape of tumors, this utility is reduced when considering small, but clinically significant sub-clones, that is, those representing <10% of the tumor burden. We have developed a high-throughput method that utilizes a 17-probe labeled bacterial artificial chromosome contig to quantify sub-clonal populations of cells based on deletion of a single locus. Chronic lymphocytic leukemia (CLL) cells harboring deletion of the short arm of chromosome 17 (del17p), an important prognostic marker for CLL were used to demonstrate the technique. Sub-clones of del17p cells were quantified and isolated from heterogeneous CLL populations using fluorescence in situ hybridization in suspension (FISH-IS) and the locus specific probe set. Using the combination of FISH-IS with the locus-specific probe set enables automated analysis of tens of thousands of cells, accurately quantifying and isolating cells carrying a del17p. Based on the fluorescence intensity of 17p probes, 17p (TP53) deleted cells were identified and sorted using flow cytometric techniques, and enrichment was demonstrated using single nucleotide polymorphism analysis. The ability to separate sub-clones of cells based on genetic heterogeneity, independent of the clone size, highlights the potential application of this method not only in the diagnostic and prognostic setting, but also as an unbiased approach to enable further detailed genetic analysis of the sub-clone with deep sequencing approaches. © 2017 International Society for Advancement of Cytometry.

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“…Next, we asked if AZ3146 treatment enhances CIN in AML cells. To do so, we performed fluorescent in situ hybridization (FISH) in cell suspension as previously described 37 . The FISH analysis revealed a baseline variance of 10% in copy number for chromosomes 6 and 8 in untreated K562 cells, suggesting that these cells exhibit basal levels of chromosome mis‐segregation.…”

Section: Resultsmentioning

confidence: 99%

Chromosomal instability upregulates interferon in acute myeloid leukemia

Jin

Lera

Yan

et al. 2020

Genes Chromosomes & Cancer

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Chromosome instability (CIN) generates genetic and karyotypic diversity that is common in hematological malignancies. Low to moderate levels of CIN are well tolerated and can promote cancer proliferation. However, high levels of CIN are lethal. Thus, CIN may serve both as a prognostic factor to predict clinical outcome and as a predictive biomarker. A retrospective study was performed to evaluate CIN in acute myeloid leukemia (AML). Chromosome mis‐segregation frequency was correlated with clinical outcome in bone marrow core biopsy specimens from 17 AML cases. Additionally, we induced chromosome segregation errors in AML cell lines with AZ3146, an inhibitor of the Mps1 mitotic checkpoint kinase, to quantify the phenotypic effects of high CIN. We observed a broad distribution of chromosome mis‐segregation frequency in AML bone marrow core specimens. High CIN correlated with complex karyotype in AML, as expected, although there was no clear survival effect. In addition to CIN, experimentally inducing chromosome segregation errors by Mps1 inhibition in AML cell lines causes DNA damage, micronuclei formation, and upregulation of interferon stimulated genes. High levels of CIN appear to be immunostimulatory, suggesting an opportunity to combine mitotic checkpoint inhibitors with immunotherapy in treatment of AML.

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Fluorescent In Situ Hybridization in Suspension by Imaging Flow Cytometry

Cited by 12 publications

References 11 publications

mRNA detection in budding yeast with single fluorophores

mRNA detection in budding yeast with single fluorophores

Development of locus specific sub‐clone separation by fluorescence in situ hybridization in suspension in chronic lymphocytic leukemia

Chromosomal instability upregulates interferon in acute myeloid leukemia

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