Journal
CHEMICAL SCIENCE
Volume 13, Issue 7, Pages 2062-2070Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/d1sc05304c
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Funding
- National Natural Science Foundation of China [21974064, 22022405, 21635005]
- Natural Science Foundation of Jiangsu Province for distinguished Young Scholars [BK20200010]
- Specially-appointed Professor Foundation of Jiangsu Province
- Program for Innovative Talents and Entrepreneurs of Jiangsu Province
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By developing a hydrogel microbead-based strategy, simultaneous sensitive quantification of multiple miRNAs in single cells is achieved. The porous structure of the hydrogel microbeads effectively avoids nonspecific cross-reactions and enables successful differentiation of cellular heterogeneity and subpopulation discrimination in liver cancer cells and normal liver cells.
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.
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