Visualization of Individual RNA Molecules by Proximity Ligation-Based Chromogenic In Situ Hybridization Assay.
Methods Mol Biol
; 2822: 143-156, 2024.
Article
in En
| MEDLINE
| ID: mdl-38907917
ABSTRACT
RNA in situ hybridization reveals the abundance and location of gene expression in cells or tissues, providing a technical basis for the clinical diagnosis of diseases. In this chapter, we show a "V" shape probe-mediated single-molecule chromogenic in situ hybridization (vsmCISH) technique for bright-field visualization of individual RNA molecules. In our method, several pairs of target hybridization probes are hybridized to RNA molecules and each probe pair forms a "V" shape overhang. The overhang oligonucleotides then mediated the proximity ligation to form DNA circles, followed by rolling circle amplification for signal enhancement and enzyme-catalyzed chromogenic reaction-based readout. The colorimetric assay avoids problems such as photobleaching and autofluorescence of current fluorescent in situ hybridization-based single-molecule RNA detection techniques. Furthermore, the relatively straightforward protocol makes the method useful for biological research and clinical diagnosis applications.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
RNA
/
In Situ Hybridization
Limits:
Humans
Language:
En
Journal:
Methods Mol Biol
/
Methods in molecular biology
/
Methods mol. biol
Journal subject:
BIOLOGIA MOLECULAR
Year:
2024
Document type:
Article
Affiliation country:
Country of publication: