Label it mirna labeling kit

NCmiR was labeled with Cy5 (^Cy5NCmiR) using LabelIT miRNA labeling kit (Mirus Bio, Madison, WI, USA). Thereafter, NPX containing Cy5NCmiR (^Cy5NCmiR:NPX-glue) or the pEGFP-C1 plasmid (pEGFP:NPX) were prepared as described above. To determine the transfection efficiency, Hep3B cells were plated at a density of 5×10⁴ cells/well on a coverslip in a 24-well plate on day 0. On day 2, ^Cy5NCmiR:NPX-glue or pEGFP:NPX-glue was placed on a side of the well and the coverslip containing the seeded cells was placed on the well. On the days indicated in the figures, ^Cy5NCmiR or GFP expression in the cells was determined using confocal fluorescence microscopy (Zeiss LSM510 Meta Confocal Imaging System, Carl Zeiss, Thornwood, NY, USA) and FACSCalibur Flow Cytometry (BD Biosciences, San Jose, CA, USA) after detaching the cells. The transfection efficiency of NPX-glue was compared with that of G-fectin (Genolution, Seoul, Korea) for miRNA and Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) for the plasmid. The cells were transfected with ^Cy5NCmiR (100 nmol) or pEGFP-C1 (0.5 μg) using G-fectin or Lipofectamine 2000, respectively, as per the manufacturer's suggestion.