Figure 7: Microinjection assay.

(a) Protocols for microinjection assay. (b,c) μCT analysis of miR-153 affected mandibular first molars. (b) 3-D reconstruction of PN12 mouse mandibular first molars. The colors varying from blue to red/yellow represents a change of enamel density from low to high. (b1–b3) miR-153: molars affected by miR-153 microinjection (right side mandibular injections); (b4–b6) Control for miR-153: left-side molars from the same animals as (b1–b3) with PBS (1X, pH 7.4) microinjection; (b7,b8) Scramble: molars affected by scrambled siRNA microinjection (right side mandibular injections); (b10–b12) Control for Scramble: left-side molar from (b10–b12) animals receiving PBS (1X, pH 7.4) microinjection. For the data presented, the concentration of miR-153 mimics and scrambled siRNA was 100 pmol. (c) Calculation and statistical analysis of relative surface enamel densities. When the miR-153 group was compared with PBS control group in the same animal, the potential differences in surface enamel densities reached statistical significance (P = 0.012). MiR-153 group showed an average of ~44% decrease in relative surface enamel density. The injection of scrambled siRNAs did not generate any statistically significant changes in surface enamel density relative to the PBS control side. Compared with scramble controls, the miR-153 affected mandibular first molars showed an average decrease of ~30% in relative enamel density (P = 0.034). (d,e) Western blot analysis of amelogenin in miR-153 injected mandibular first molars. (d) The target band of amelogenin was at 20-kD. Beta-actin was used to control the amount of sample loading. (e) The densities of target band were measured relative to beta-actin. Statistically significant differences were detected between miR-153 microinjection group (miR-153) and PBS control group (Control for miR-153), and between scrambled siRNA microinjection group (Scramble) and corresponding PBS control group (Control for scramble). *P < 0.05.