Native FISH: A low- and high-throughput assay to analyze the alternative lengthening of telomere (ALT) pathway.
Methods Cell Biol
; 182: 265-284, 2024.
Article
en En
| MEDLINE
| ID: mdl-38359982
ABSTRACT
Alternative lengthening of telomeres (ALT) is a telomerase-independent and recombination-based mechanism used by approximately 15% of human cancers to maintain telomere length and to sustain proliferation. ALT-positive cells display unique features that could be exploited for tailored cancer therapies. A key limitation for the development of ALT-specific treatments is the lack of an assay to detect ALT-positive cells that is easy to perform and that can be scaled up. One of the most broadly used assays for ALT detection, CCA (C-circle assay), does not provide single-cell information and it is not amenable to High-Throughput Screening (HTS). To overcome these limitations, we developed Native-FISH (N-FISH) as an alternative method to visualize ALT-specific single-stranded telomeric DNA. N-FISH produces single-cell data, can be applied to fixed tissues, does not require DNA isolation or amplification steps, and it can be miniaturized in a 384-well format. This protocol details the steps to perform N-FISH protocol both in a low- and high-throughput format to analyze ALT. While low-throughput N-FISH is useful to assay the ALT state of cell lines, we expect that the miniaturized N-FISH assay coupled with high-throughput imaging will be useful in functional genomics and chemical screens to identify novel cellular factors that regulate ALT and potential ALT therapeutic targets for cancer therapies directed against ALT-positive tumors, respectively.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Ensayos Analíticos de Alto Rendimiento
/
Neoplasias
Tipo de estudio:
Guideline
/
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Methods Cell Biol
Año:
2024
Tipo del documento:
Article
Pais de publicación:
Estados Unidos