Detection of MicroRNA Expression Dynamics Using LNA/DNA Nanobiosensor.
Methods Mol Biol
; 2630: 75-87, 2023.
Article
em En
| MEDLINE
| ID: mdl-36689177
ABSTRACT
The investigation of complex biological processes requires effective tools for probing the spatiotemporal dynamics of individual cells. Single-cell gene expression analysis, such as RNA in situ hybridization and single-cell PCR, has been demonstrated in various biological applications (Tautz and Pfeifle, Chromosoma 98(2)81-5, 1989; Stahlberg and Bengtsson, Methods 50(4)282-288, 2010; Sanchez-Freire et al., Nat Protoc 7(5)829-838, 2012). However, existing techniques require cell lysis or fixation. The dynamic information and spatiotemporal regulation of the biological process cannot be obtained with these methods. Real-time gene expression analysis in living cells remains an outstanding challenge in the field. Here, we described a single-cell gene expression analysis method in living mammalian cells using a locked nucleic acid/DNA (LNA/DNA) nanobiosensor. This LNA/DNA nanobiosensor consists of a fluorophore-labeled detecting strand and a quenching strand. The fluorophore-labeled LNA probe is designed to hybridize with the target microRNA (miRNA) specifically and displace from the quenching strand, allowing the fluorophore to fluorescence. Large-scale single-cell dynamic gene expression monitoring can be performed using time-lapse microscopy to study spatiotemporal distribution and heterogeneity in gene expression. Multiplex detection of miRNAs can be achieved using different fluorophore-labeled LNA/DNA nanobiosensors. This LNA/DNA protocol is fast, generally applicable, and easily accessible.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA
/
MicroRNAs
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
Idioma:
En
Revista:
Methods Mol Biol
Ano de publicação:
2023
Tipo de documento:
Article