The precise identification and separation of living cell types is critical to both study cell function and prepare cells for medical applications. achievable. Our method could identify target live cells and improve the efficiency of cell purification from heterogeneous populations. Multicellular organisms consist of many cell types each with different function1. The identification and separation of target living cells is critical not only to reveal their function but also to use them for therapeutic purposes. Marker proteins on the cell surface are commonly used to identify living cell types2 3 4 5 However obtaining specific sets of surface markers and their corresponding antibodies is not trivial. Moreover these antibody-detected cell populations often remain heterogeneous and composed of subgroups that cannot be distinguished to each other6. To improve the resolution of live cell identification and separation means for quantitative detection and utilization of intracellular markers are Rabbit Polyclonal to IRX2. essential. MicroRNAs (miRNAs) which are short non-coding RNAs transcribed in plants and animals7 8 are a Etoricoxib potential non-protein intracellular biomarker to distinguish cell types9 10 The nature of nucleic acids is suitable for quantitative measurement in a high-throughput manner11 12 13 despite difficulty in applying such methods to live cells. The activity of miRNA Etoricoxib is controlled during various biological processes14. Thus Etoricoxib the measurement of miRNA activity rather than that of miRNA quantity is more suitable for live cell identification and separation. We have recently reported a method to purify cell populations using synthetic mRNA that detects highly expressed miRNA in target cells15. However identification and separation of cells with similar miRNA profiles remains a challenge though it is particularly important for the quality control of cells in therapeutic applications. In this study 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 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 Etoricoxib 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. A single miRNA target site which is completely complementary to the Etoricoxib target miRNA in the 5′ UTR of synthetic mRNAs efficiently detected miRNA activity in a cell15. Then we assumed that the combination of two distinct miRNA-responsive mRNAs can improve the resolution for cell identification: If two miRNAs are more and less active respectively in one cell type compared with the other cell type then subtle differences in miRNA activities should be sufficient to distinguish them (Fig. 1). 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). Figure 1 Schematic illustration of the high-resolution identification and separation of cell types. Results To demonstrate our HRIC concept we arbitrarily selected 20 miRNAs that have previously been examined in HeLa cells (human cervical cancer cells)16 and tested their activity in HeLa MCF-7 (human breast cancer cells) and 293FT cells (human embryo kidney cells) using the 20 miRNA-responsive mRNAs that encode enhanced green fluorescent protein (EGFP) (Supplementary Table S1). We compared the miRNA activities between HeLa and MCF-7 cells and found no miRNAs whose activity differed more than two-fold (Fig. 2a). Thus we used HeLa and MCF-7 cells as a model case Etoricoxib to distinguish cells with similar miRNA profiles. We investigated whether the cotransfection of two distinct miRNA-responsive mRNAs could separate HeLa and MCF-7 cells based on less than two-fold differences in the miRNA activities. We chose miR-24-3p and miR-203a which were 1.3-fold more active and 1.5-fold less active in HeLa than MCF-7 cells respectively (Fig. 2b). The cotransfection of these two miRNA-responsive mRNAs.