Allele-level visualization of transcription and chromatin by high-throughput imaging.
DNA/RNA FISH
Genome organization
High-throughput imaging
In situ hybridization
Journal
Histochemistry and cell biology
ISSN: 1432-119X
Titre abrégé: Histochem Cell Biol
Pays: Germany
ID NLM: 9506663
Informations de publication
Date de publication:
09 May 2024
09 May 2024
Historique:
accepted:
11
04
2024
medline:
10
5
2024
pubmed:
10
5
2024
entrez:
9
5
2024
Statut:
aheadofprint
Résumé
The spatial arrangement of the genome within the nucleus is a pivotal aspect of cellular organization and function with implications for gene expression and regulation. While all genome organization features, such as loops, domains, and radial positioning, are nonrandom, they are characterized by a high degree of single-cell variability. Imaging approaches are ideally suited to visualize, measure, and study single-cell heterogeneity in genome organization. Here, we describe two methods for the detection of DNA and RNA of individual gene alleles by fluorescence in situ hybridization (FISH) in a high-throughput format. We have optimized combined DNA/RNA FISH approaches either using simultaneous or sequential detection of DNA and nascent RNA. These optimized DNA and RNA FISH protocols were implemented in a 384-well plate format alongside automated image and data analysis and enable accurate detection of individual gene alleles and their gene expression status across a large cell population. We successfully visualized MYC and EGFR DNA and nascent RNA with allele-level resolution in multiple cell types, and we determined the radial position of active and inactive MYC and EGFR alleles. These optimized DNA/RNA detection approaches are versatile and sensitive tools for mapping of chromatin features and gene activity at the single-allele level and at high throughput.
Identifiants
pubmed: 38724854
doi: 10.1007/s00418-024-02289-7
pii: 10.1007/s00418-024-02289-7
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : NIH HHS
ID : 1-ZIA-BC010309-24
Pays : United States
Organisme : NIH HHS
ID : 1-ZIC-BC011567-10
Pays : United States
Organisme : NIH HHS
ID : 1-ZIC-BC011567-10
Pays : United States
Organisme : NIH HHS
ID : 1-ZIA-BC010309-24
Pays : United States
Informations de copyright
© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.
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