Wang Yingfei, Fang Yanyun, Zhu Yu, Bi Shiyi, Liu Ying, Ju Huangxian
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University Nanjing 210023 PR China.
Chemistry and Biomedicine Innovation Center, Nanjing University Nanjing 210023 China
Chem Sci. 2022 Jan 27;13(7):2062-2070. doi: 10.1039/d1sc05304c. eCollection 2022 Feb 16.
The simultaneous quantification of multi-miRNAs in single cells reveals cellular heterogeneity, and benefits the subtypes discrimination of cancer cells . Though micro-droplet techniques enable successful single cell encapsulation, the isolated and restricted reaction space of microdroplets causes cross-reactions and inaccuracy for simultaneous multi-miRNAs quantification. Herein, we develop a hydrogel microbead based strategy for the simultaneous sensitive quantification of miRNA-21, 122 and 222 in single cells. Single cells are encapsulated and undergo cytolysis in hydrogel microbeads. The three target miRNAs are retained in the microbead by pre-immobilized capture probes, and activate rolling circle amplification (RCA) reactions. The RCA products are hybridized with corresponding dye labelled DNA reporters, and the respective fluorescence intensities are recorded for multi-miRNA quantification. The porous structure of the hydrogel microbeads allows the free diffusion of reactants and easy removal of unreacted DNA strands, which effectively avoids nonspecific cross-reactions. Clear differentiation of cellular heterogeneity and subpopulation discrimination are achieved for three kinds of liver cancer cells and one normal liver cell.
单细胞中多种微小RNA的同时定量揭示了细胞异质性，并有助于癌细胞亚型的区分。尽管微滴技术能够成功地实现单细胞封装，但微滴孤立且受限的反应空间会导致交叉反应以及同时进行多种微小RNA定量时的不准确。在此，我们开发了一种基于水凝胶微珠的策略，用于在单细胞中同时灵敏地定量微小RNA-21、122和222。单细胞被封装在水凝胶微珠中并进行细胞裂解。三种靶标微小RNA通过预固定的捕获探针保留在微珠中，并激活滚环扩增（RCA）反应。RCA产物与相应的染料标记DNA报告分子杂交，并记录各自的荧光强度以进行多微小RNA定量。水凝胶微珠的多孔结构允许反应物自由扩散，并易于去除未反应的DNA链，这有效地避免了非特异性交叉反应。对于三种肝癌细胞和一种正常肝细胞，实现了细胞异质性的清晰区分和亚群区分。
