Single cell multi-miRNAs quantification with hydrogel microbeads for liver cancer cell subtypes discrimination

Area of Science:

• Biotechnology
• Molecular Biology
• Single-cell analysis

Background:

• Simultaneous quantification of multiple microRNAs (miRNAs) in single cells is crucial for understanding cellular heterogeneity and cancer subtypes.
• Micro-droplet techniques, while enabling single-cell encapsulation, suffer from restricted reaction spaces leading to cross-reactions and inaccurate multi-miRNA quantification.

Purpose of the Study:

• To develop a novel hydrogel microbead-based strategy for sensitive and simultaneous quantification of specific miRNAs (miRNA-21, 122, and 222) in single cells.
• To overcome the cross-reaction and inaccuracy issues associated with micro-droplet methods for multi-miRNA detection.

Main Methods:

• Single cells are encapsulated within hydrogel microbeads, followed by cell lysis.
• Pre-immobilized capture probes within the microbeads retain target miRNAs and initiate rolling circle amplification (RCA).
• RCA products are hybridized with fluorescently labeled DNA reporters for sensitive, quantitative detection of multiple miRNAs.

Main Results:

• The hydrogel microbead strategy effectively prevents nonspecific cross-reactions due to its porous structure, allowing free diffusion and removal of unreacted reagents.
• Simultaneous and sensitive quantification of miRNA-21, 122, and 222 in single cells was achieved.
• The method successfully differentiated cellular heterogeneity and discriminated subpopulations among three types of liver cancer cells and one normal liver cell type.

Conclusions:

• Hydrogel microbeads offer a robust platform for accurate single-cell multi-miRNA analysis, surpassing micro-droplet limitations.
• This technique provides a powerful tool for dissecting cellular heterogeneity and aiding in cancer subtyping.
• The developed method enables sensitive and specific detection, paving the way for advanced single-cell diagnostics.