title: Molecular Beacon-Based MicroRNA Imaging During Neurogenesis.
abstract: The fluorescence monitoring system for examining endogenous microRNA (miRNA) activity in cellular level provides crucial information on not only understanding a critical role of miRNA involving a variety of biological processes, but also evaluating miRNA expression patterns in a noninvasive manner. In this protocol, we report the details of a new procedure for a molecular beacon-based miRNA monitoring system, which includes the illustration scheme for miRNA detection strategy, exogenous miRNA detection, and measurement of endogenous miRNA expression level during neurogenesis. The fluorescence signal of miR-124a beacon quenched by BHQ2 was gradually recovered as increasing concentration of the miR-124a in tube. The functional work of miR-124a beacon was examined in intracellular environment, allowing for the internalization of the miR-124a beacon by lipofectamine, which resulted in activated fluorescent signals of the miR-124a beacon in the HeLa cells after the addition of synthetic miR-124a. The endogenous miR-124a expression level was detected by miR-124a beacon system during neurogenesis, showing brighter fluorescence intensity in cytoplasmic area of P19 cells after induction of neuronal differentiation by retinoic acid. The molecular beacon based-miRNA detection technique could be applicable to the simultaneous visualization of a variety of miRNA expression patterns using different fluorescence dyes. For the study of examining endogenous miRNA expression level using miRNA-beacon system, if cellular differentiation step is already prepared, transfection step of miR-124a beacon into P19 cells, and acquisition of activated fluorescence signal measured by confocal microscope can be conducted approximately within 6 h.
authors.affiliation: Institute for Bio-Medical Convergence, College of Medicine, Catholic Kwandong University, Gangneung-si, Gangwon-do, 270-701, Republic of Korea.|Catholic Kwandong University International St. Mary's Hospital, Incheon Metropolitan City, 404-834, Republic of Korea.
authors.forename: Jonghwan
authors.initials: J
authors.lastname: Lee
authors.affiliation: Institute for Bio-Medical Convergence, College of Medicine, Catholic Kwandong University, Gangneung-si, Gangwon-do, 270-701, Republic of Korea. kimsoonhag@empal.com.|Catholic Kwandong University International St. Mary's Hospital, Incheon Metropolitan City, 404-834, Republic of Korea. kimsoonhag@empal.com.
authors.forename: Soonhag
authors.initials: S
authors.lastname: Kim
mesh_terms: D002454:Cell Differentiation; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods; D057054:Molecular Imaging / Q000379:methods*; D055495:Neurogenesis / Q000235:genetics*; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes; C509207:MIRN124 microRNA, human; D035683:MicroRNAs
keywords: Imaging; Molecular beacon; Neurogenesis; microRNA