Multiplex miRNA reporting platform for real-time profiling of living cells.

Accurately characterizing cell types within complex cell structures provides invaluable information for comprehending the cellular status during biological processes. In this study, we have developed an miRNA-switch cocktail platform capable of reporting and tracking the activities of multiple miRNAs (microRNAs) at the single-cell level, while minimizing disruption to the cell culture. Drawing on the principles of traditional miRNA-sensing mRNA switches, our platform incorporates subcellular tags and employs intelligent engineering to segment three subcellular regions using two fluorescent proteins. These designs enable the quantification of multiple miRNAs within the same cell. Through our experiments, we have demonstrated the platform's ability to track marker miRNA levels during cell differentiation and provide spatial information of heterogeneity on outlier cells exhibiting extreme miRNA levels. Importantly, this platform offers real-time and in situ miRNA reporting, allowing for multidimensional evaluation of cell profile and paving the way for a comprehensive understanding of cellular events during biological processes. [Display omitted] • Multiplex miRNA reporting that goes beyond the fluorescent channel limit using subcellular tags • Subcellular area segmentation using reduced number of fluorescent channels • Multidimensional and single-cell level miRNA reporting for in-depth cell profiling Hu et al. present a microRNA reporting platform that can quantify multiple miRNAs at real time for profiling of living cells. This platform enables simultaneous tracking of the dynamic change of related miRNAs during biological processes and spatial cell-cell miRNA heterogeneity. [ABSTRACT FROM AUTHOR]