电子化试验记录及相关信息分享工具需求调研问卷导师-Bio-protocol.PDFVIP

Vol 8, Iss 17, Sep 05, 2018 /e3000 DOI:10.21769/BioProtoc.3000 Dual Fluorescence Reporter Based Analytical Flow Cytometry for miRNA Induced Regulation in Mammalian Cells Nicolas Lemus-Diaz*, Liezel Tamon and Jens Gruber* Junior Research Group Medical RNA Biology, German Primate Center, Leibniz Institute for Primate Research, Kellnerweg 4, G枚ttingen, Germany *For correspondence: Nicolas.lemus@; jgruber@dpz.eu [Abstract] MicroRNA-induced gene regulation is a growing field in basic and translational research. Examining this regulation directly in cells is necessary to validate high-throughput data originated from RNA sequencing technologies. For this several studies employ luciferase-based reporters that usually measure the whole cell population, which comes with low resolution for the complexity of the miRNA-induced regulation. Here, we provide a protocol using a dual-fluorescence reporter and flow cytometry reaching single cell resolution; the protocol contains a simplified workflow that includes: vector generation, data acquisition, processing, and analysis using the R environment. Our protocol enables high-resolution measurements of miRNA induced post-transcriptional gene regulation and combined with system biology it can be used to estimate miRNAs proficiency. Keywords: miRNA, Flow-Cytometry, Dual fluorescence reporter, Functional assay, ncRNA, Small RNA, non-protein-coding RNA, Gene regulation, Reporter gene system, Single cell analysis, Flow cytometry [Background] MicroRNAs (miRNA) are highly conserved small non-protein-coding RNAs (21-22 nt) that regulate post-transcriptionally gene expression and modulate fundamental bio