Single molecule chromogenic in situ hybridization assay for RNA visualization in fixed cells and tissues.
Meng JiangLing LiuChengye HongDebo ChenXihu YaoXiaoyuan ChenChen LinRongqin KePublished in: RNA (New York, N.Y.) (2019)
Visualization of gene expression at single RNA molecular level represents a great challenge to both imaging technologies and molecular engineering. Here we show a single molecule chromogenic in situ hybridization (smCISH) assay that enables counting and localizing individual RNA molecules in fixed cells and tissue under bright-field microscopy. Our method is based on in situ padlock probe assays directly using RNA as a ligation template and rolling circle amplification combined with enzyme catalyzed chromogenic reaction for amplification product visualization. We show potential applications of our method by detecting gene expression variations in single cells, subcellular localization information of expressed genes, and gene expression heterogeneity in formalin-fixed, paraffin-embedded tissue sections. This facile and straightforward method can in principle be applied to any type of RNA molecules in different samples.
Keyphrases
- single molecule
- gene expression
- induced apoptosis
- nucleic acid
- living cells
- cell cycle arrest
- dna methylation
- atomic force microscopy
- high throughput
- high resolution
- signaling pathway
- oxidative stress
- cell proliferation
- genome wide
- molecularly imprinted
- genome wide identification
- room temperature
- bioinformatics analysis
- label free
- solid phase extraction