High-resolution Identification and Separation of Living Cell Types by Multiple microRNA-responsive Synthetic mRNAs.

Kei Endo,Karin Hayashi,Hirohide Saito

doi:10.1038/srep21991

Abstract

The precise identification and separation of living cell types is critical to both study cell function and prepare cells for medical applications. However, intracellular information to distinguish live cells remains largely inaccessible. Here, we develop a method for high-resolution identification and separation of cell types by quantifying multiple microRNA (miRNA) activities in live cell populations. We found that a set of miRNA-responsive, in vitro synthesized mRNAs identify a specific cell population as a sharp peak and clearly separate different cell types based on less than two-fold differences in miRNA activities. Increasing the number of miRNA-responsive mRNAs enhanced the capability for cell identification and separation, as we precisely and simultaneously distinguished different cell types with similar miRNA profiles. In addition, the set of synthetic mRNAs separated HeLa cells into subgroups, uncovering heterogeneity of the cells and the level of resolution achievable. Our method could identify target live cells and improve the efficiency of cell purification from heterogeneous populations.

Highlights

MicroRNAs, which are short non-coding RNAs transcribed in plants and animals[7,8] are a potential non-protein intracellular biomarker to distinguish cell types[9,10]
We developed a means for the high-resolution identification of cell types (HRIC) based on the simultaneous quantification of multiple miRNA activities in live cells using a set of miRNA-responsive, synthetic mRNAs
Because we found that the peak width of the fluorescence ratios translated from two reporter mRNAs tends to distribute within four-fold, using our strategy, less than two-fold differences in two miRNA activities, which result in approximately four-fold difference in the fluorescence ratios, is sufficient to distinguish two cell types (Fig. 1, bottom)

Summary

Introduction

MicroRNAs (miRNAs), which are short non-coding RNAs transcribed in plants and animals[7,8] are a potential non-protein intracellular biomarker to distinguish cell types[9,10]. We developed a means for the high-resolution identification of cell types (HRIC) based on the simultaneous quantification of multiple miRNA activities in live cells using a set of miRNA-responsive, synthetic mRNAs. Transfection of two in vitro synthesized mRNAs that encode different fluorescent proteins (FL1 or FL2) enabled us to detect a specific cell population as a peak (Supplementary Fig. S1), because the fluorescence ratio of the reporter proteins from the two mRNAs (FL2/FL1) was almost constant irrespective of the transferred mRNA levels in each cell.

Results

Conclusion