Figure 2.

Confirmation of maturation of introduced miRNAs in single living cells

(A) Time courses of the normalized amplitude of fluorescence cross-correlation functions (FCFs) of fluorescently labeled guide and passenger strands of let-7a-1 pre-miRNA (left) and pre-miRNA of miR-21 (right) in the cytoplasm. The decay curves of FCFs were approximated with an exponential decay with time constants of 43.4 min (let-7a-1) and 36.3 min (miR-21). Error bars represent SD (n = 5 cells).

(B) Colocalization of let-7a-1 pre-miRNA (left), its target Kras mRNA antisense probes (middle), and their merged (right) image. Trajectories of extracted foci in which (pre-)miRNA and mRNA antisense probe(s) co-localized are shown (far right). White circles indicate the example of the colocalization of let-7a-1 (pre-) miRNA and Kras mRNA.

(C) Fluorescence images of let-7a-1 pre-miRNA (guide strand was labeled with 2MeSiR, left) and Luciferase mRNA antisense probe (labeled with Cy3B, middle), and their merged (right) image. No co-localized focus was extracted (far right).

(B and C) Representative single-frame images of let-7a-1 and mRNA probes at a given time are shown. White scale bars indicate 10 μm.

(D) Localization of the specific miRNA in exosome. Epi-fluorescence images of exosome visualized with CD63-GFP (left), pre-miRNAs (middle), and their merged images (right). miRNA-198 localized in exosomes, while let-7a-1 miRNA did not. The regions shown in the squares in the lower panels are enlarged in insets above them to the right in the panels. White scale bars indicate 20 μm.