Single molecule chromogenic in situ hybridization assay for RNA visualization in fixed cells and tissues. 2019

Meng Jiang, and Ling Liu, and Chengye Hong, and Debo Chen, and Xihu Yao, and Xiaoyuan Chen, and Chen Lin, and Rongqin Ke
Center for Precision Medicine, School of Biomedical Sciences and School of Medicine, Huaqiao University, Quanzhou, Fujian, 362021, China.

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.

UI MeSH Term Description Entries
D002863 Chromogenic Compounds Colorless, endogenous or exogenous pigment precursors that may be transformed by biological mechanisms into colored compounds; used in biochemical assays and in diagnosis as indicators, especially in the form of enzyme substrates. Synonym: chromogens (not to be confused with pigment-synthesizing bacteria also called chromogens). Chromogenic Compound,Chromogenic Substrate,Chromogenic Substrates,Compound, Chromogenic,Compounds, Chromogenic,Substrate, Chromogenic,Substrates, Chromogenic
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000072760 Single Molecule Imaging High resolution imaging techniques that allow visualization of individual molecules of proteins, lipids, or nucleic acids within cells or tissues. Single Molecule Analysis,Single Molecule Tracking,Single Particle Analysis,Single Particle Imaging,Single Particle Microscopy,Single Particle Spectroscopy,Single Particle Tracking,Single Molecule Microscopy,Single Molecule Spectroscopy,Analyses, Single Particle,Analysis, Single Molecule,Analysis, Single Particle,Imaging, Single Molecule,Imaging, Single Particle,Microscopies, Single Particle,Microscopy, Single Molecule,Microscopy, Single Particle,Particle Tracking, Single,Single Molecule Analyses,Single Particle Analyses,Single Particle Microscopies,Single Particle Spectroscopies,Single Particle Trackings,Spectroscopy, Single Molecule,Spectroscopy, Single Particle,Tracking, Single Molecule,Tracking, Single Particle
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D012333 RNA, Messenger RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm. Messenger RNA,Messenger RNA, Polyadenylated,Poly(A) Tail,Poly(A)+ RNA,Poly(A)+ mRNA,RNA, Messenger, Polyadenylated,RNA, Polyadenylated,mRNA,mRNA, Non-Polyadenylated,mRNA, Polyadenylated,Non-Polyadenylated mRNA,Poly(A) RNA,Polyadenylated mRNA,Non Polyadenylated mRNA,Polyadenylated Messenger RNA,Polyadenylated RNA,RNA, Polyadenylated Messenger,mRNA, Non Polyadenylated
D015870 Gene Expression The phenotypic manifestation of a gene or genes by the processes of GENETIC TRANSCRIPTION and GENETIC TRANSLATION. Expression, Gene,Expressions, Gene,Gene Expressions
D016610 Tissue Embedding The technique of placing cells or tissue in a supporting medium so that thin sections can be cut using a microtome. The medium can be paraffin wax (PARAFFIN EMBEDDING) or plastics (PLASTIC EMBEDDING) such as epoxy resins. Embedding, Tissue
D016707 Tissue Fixation The technique of using FIXATIVES in the preparation of cytologic, histologic, or pathologic specimens for the purpose of maintaining the existing form and structure of all the constituent elements. Fixation, Tissue
D017404 In Situ Hybridization, Fluorescence A type of IN SITU HYBRIDIZATION in which target sequences are stained with fluorescent dye so their location and size can be determined using fluorescence microscopy. This staining is sufficiently distinct that the hybridization signal can be seen both in metaphase spreads and in interphase nuclei. FISH Technique,Fluorescent in Situ Hybridization,Hybridization in Situ, Fluorescence,FISH Technic,Hybridization in Situ, Fluorescent,In Situ Hybridization, Fluorescent,FISH Technics,FISH Techniques,Technic, FISH,Technics, FISH,Technique, FISH,Techniques, FISH

Related Publications

Meng Jiang, and Ling Liu, and Chengye Hong, and Debo Chen, and Xihu Yao, and Xiaoyuan Chen, and Chen Lin, and Rongqin Ke
January 2023, Methods in molecular biology (Clifton, N.J.),
Meng Jiang, and Ling Liu, and Chengye Hong, and Debo Chen, and Xihu Yao, and Xiaoyuan Chen, and Chen Lin, and Rongqin Ke
February 2023, Microbiology spectrum,
Meng Jiang, and Ling Liu, and Chengye Hong, and Debo Chen, and Xihu Yao, and Xiaoyuan Chen, and Chen Lin, and Rongqin Ke
January 2022, Methods in molecular biology (Clifton, N.J.),
Meng Jiang, and Ling Liu, and Chengye Hong, and Debo Chen, and Xihu Yao, and Xiaoyuan Chen, and Chen Lin, and Rongqin Ke
March 2024, ACS nano,
Meng Jiang, and Ling Liu, and Chengye Hong, and Debo Chen, and Xihu Yao, and Xiaoyuan Chen, and Chen Lin, and Rongqin Ke
January 2015, Methods in molecular biology (Clifton, N.J.),
Meng Jiang, and Ling Liu, and Chengye Hong, and Debo Chen, and Xihu Yao, and Xiaoyuan Chen, and Chen Lin, and Rongqin Ke
November 2018, Bio-protocol,
Meng Jiang, and Ling Liu, and Chengye Hong, and Debo Chen, and Xihu Yao, and Xiaoyuan Chen, and Chen Lin, and Rongqin Ke
February 2020, Bio-protocol,
Meng Jiang, and Ling Liu, and Chengye Hong, and Debo Chen, and Xihu Yao, and Xiaoyuan Chen, and Chen Lin, and Rongqin Ke
January 2024, Methods in molecular biology (Clifton, N.J.),
Meng Jiang, and Ling Liu, and Chengye Hong, and Debo Chen, and Xihu Yao, and Xiaoyuan Chen, and Chen Lin, and Rongqin Ke
April 2023, Journal of experimental botany,
Meng Jiang, and Ling Liu, and Chengye Hong, and Debo Chen, and Xihu Yao, and Xiaoyuan Chen, and Chen Lin, and Rongqin Ke
November 2013, Journal of visualized experiments : JoVE,
Copied contents to your clipboard!