Figure 1. Molecular beacons are new, specific tools to detect endogenous pre‐miRNAs in cells.

• A
  Schematic representation of the experimental protocol. LCM, laser capture microdissection.
• B, C
  RT–PCR performed on RNA extracted from axons collected by LCM (Ax) or from eyes. (B) β‐Actin mRNA is present both in eyes and in axons, while MAP2 and H4 are present only in the eye sample, suggesting the absence of contamination from cell bodies or other cells in LCM axonal samples (Bellon et al, 2017). Ax, axonal sample; Eye, stage 37/38 eye; LCM, laser capture microdissection; –, PCR no template control; NT, RT no template control.
• D
  Schematic of MB, pre‐miR‐181a‐1, and their hybridization complex. MB, molecular beacon.
• E
  Schematic of thermal denaturation profile of the MB. MB, molecular beacon.
• F
  Thermal denaturation profiles of MB, in the absence (solid line) and presence (dashed line) of increasing target concentration. Each melting curve represents the average of three separate replicates. Yellow boxes indicate the range of working temperatures for ex vivo trafficking experiments. MB, molecular beacon; T _m, MB melting temperature.
• G
  Schematic representation of the experimental protocol. Concentrations used are as follows: 5 μM MB; 250 μM co‐MO; 250 μM pri‐miR‐MO. MB, molecular beacon; co‐MO, control morpholino; pri‐miR‐MO, morpholino blocking pre‐miR‐181a‐1 processing by targeting the Drosha cleavage site.
• H
  Quantification of the expression levels of pre‐mir‐181a‐1 using the 2^−ΔCt method and U6 as normalizer from small total RNA fraction (< ˜150 nt). Each data point corresponds to one independent experiment. n = 3 independent experiments. Values are mean ± SEM. ns, not significant.
• I
  Total number of MB puncta normalized to axon length (μm). Each data point corresponds to one axon. Total number of puncta and axons analyzed is as follows: 928 puncta and 61 axons (WT); 226 puncta and 15 axons (co‐MO); 208 puncta and 35 axons (MO). n = 4 independent experiments. Values are mean ± SEM. ns, not significant; co‐MO, control morpholino; pri‐miR‐MO, morpholino blocking pre‐miR‐181a‐1 processing by targeting the Drosha cleavage site.
• J
  Representative axons. MB puncta are indicated (white arrows). Dashed white lines delineate axons. MB, molecular beacon. Scale bars: 5 μm.
• K
  Schematic representation of the experimental protocol. Concentrations used are as follows: 5 μM MB; 200–250 ng/μl cy3‐pre‐miR‐181a‐1. MB, molecular beacon.
• M
  Schematic representation of cy5‐labeled pre‐miR‐181a‐1.
• N
  Frequency (in percentage) of puncta colocalization between MB and cy5‐pre‐miR‐181a‐1 (#MB+/pre‐miR+). Each data point corresponds to one axon. Total number of puncta and axons analyzed is as follows: 354 puncta and 32 axons (MB); 337 puncta and 32 axons (cy5‐pre‐miR‐181a‐1). n = 5 independent experiments. Values are mean ± SEM. MB, molecular beacon.
• O
  Representative image of RGC axons. White, red, and blue arrows indicate, respectively, colocalized, single MB, and single pre‐miR‐181a‐1 puncta. Scale bars: 5 μm.

Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data were normally distributed (Shapiro–Wilk test), one‐way ANOVA followed by Tukey's multiple comparison post hoc test (H). Data were not normally distributed (Shapiro–Wilk test), and Kruskal–Wallis test followed by Dunn's multiple comparison post hoc test (I).