In Situ Imaging of Individual mRNA Mutation in Single Cells Using Ligation-Mediated Branched Hybridization Chain Reaction (Ligation-bHCR),Analytical Chemistry

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In Situ Imaging of Individual mRNA Mutation in Single Cells Using Ligation-Mediated Branched Hybridization Chain Reaction (Ligation-bHCR)
Analytical Chemistry ( IF 6.7 ) Pub Date : 2017-03-06 00:00:00 , DOI: 10.1021/acs.analchem.6b04312
Ying Tang ₁ , Xiao-Li Zhang ₁ , Li-Juan Tang ₁ , Ru-Qin Yu ₁ , Jian-Hui Jiang ₁

Affiliation

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.

中文翻译：

使用连接介导的分支杂交链反应（Ligation-bHCR）原位成像单个细胞中的单个mRNA突变

基因表达的超灵敏和特异性原位成像对于分子医学和临床治疗学至关重要。我们开发了一种基于新的分支杂交链反应（bHCR）的新型荧光原位杂交（FISH）策略，用于在FISH分析中进行有效的信号放大和连接酶介导的特异性突变检测。据我们所知，这是首次在FISH分析中实现用于突变检测的HCR。体外测定表明，连接-bHCR策略在区分mRNA中的单核苷酸变异方面具有很高的特异性，并且可产生高度分支的聚合产物，与HCR相比，该产物具有更高的扩增效率或更高的灵敏度。成像分析表明，连接-bHCR产生高亮度的斑点样信号，用于单个mRNA分子的定位，而不同颜色的斑点信号在单个mRNA的基因分型点突变中具有高度特异性。此外，该策略显示具有对单细胞水平上的mRNA表达进行定量成像的潜力。因此，该策略可为开发用于各种诊断和研究应用的高度敏感和特定的FISH方法提供新的有希望的范例。

更新日期：2017-03-06

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