title: In Situ Imaging of Individual mRNA Mutation in Single Cells Using Ligation-Mediated Branched Hybridization Chain Reaction (Ligation-bHCR).
abstract: Ultrasensitive and specific in situ imaging of gene expression is essential for molecular medicine and clinical theranostics. We develop a novel fluorescence in situ hybridization (FISH) strategy based on a new branched hybridization chain reaction (bHCR) for efficient signal amplification in the FISH assay and a ligase-mediated discrimination for specific mutation detection. To our knowledge, this is the first time that HCR has been realized for mutation detection in the FISH assay. In vitro assay shows that the ligation-bHCR strategy affords high specificity in discriminating single-nucleotide variation in mRNA, and it generates a highly branched polymeric product that confers more efficient amplification or better sensitivity than HCR. Imaging analysis reveals that ligation-bHCR generates highly bright spot-like signals for localization of individual mRNA molecules, and spot signals of different colors are highly specific in genotyping point mutation of individual mRNA. Moreover, this strategy is shown to have the potential for quantitative imaging of the expression of mRNA at the single-cell level. Therefore, this strategy may provide a new promising paradigm in developing highly sensitive and specific FISH methods for various diagnostic and research applications.
authors.affiliation: State Key Laboratory of Chemeo/Bio-Sensing and Chemometrics, Institute of Chemical Biology and Nanomedicine, College of Chemistry and Chemical Engineering, Hunan University , Changsha 410082, P. R. China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Tang
authors.affiliation: State Key Laboratory of Chemeo/Bio-Sensing and Chemometrics, Institute of Chemical Biology and Nanomedicine, College of Chemistry and Chemical Engineering, Hunan University , Changsha 410082, P. R. China.
authors.forename: Xiao-Li
authors.initials: XL
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Chemeo/Bio-Sensing and Chemometrics, Institute of Chemical Biology and Nanomedicine, College of Chemistry and Chemical Engineering, Hunan University , Changsha 410082, P. R. China.
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Tang
authors.affiliation: State Key Laboratory of Chemeo/Bio-Sensing and Chemometrics, Institute of Chemical Biology and Nanomedicine, College of Chemistry and Chemical Engineering, Hunan University , Changsha 410082, P. R. China.
authors.forename: Ru-Qin
authors.identifier: 0000-0002-7412-8360
authors.initials: RQ
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Chemeo/Bio-Sensing and Chemometrics, Institute of Chemical Biology and Nanomedicine, College of Chemistry and Chemical Engineering, Hunan University , Changsha 410082, P. R. China.
authors.forename: Jian-Hui
authors.identifier: 0000-0003-1594-4023
authors.initials: JH
authors.lastname: Jiang
mesh_terms: D019073:HT29 Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008025:Ligases / Q000737:chemistry / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D009693:Nucleic Acid Hybridization; D061848:Optical Imaging; D012333:RNA, Messenger / Q000235:genetics*; D059010:Single-Cell Analysis; D014407:Tumor Cells, Cultured
chemical_list: D012333:RNA, Messenger; D008025:Ligases