Mirna probes

Total RNA (8–20 μg) from 21 hpf embryos and adults were isolated using RNAiso Reagent (Takara) and electrophoresed in a denaturing 12% polyacrylamide gel and transferred onto a Hybond-N+ membrane (GE Healthcare). miRNA probes (Additional file 5: Table S3) were DIG-labeled LNA-modified probes synthesized by Exiqon (Denmark). Hybridization and washing were performed as described by Válóczi [36 (link)]. Signals were detected using CDP-Star chemiluminescent substrate (Roche), and the blot was exposed to the Lumi-Imager F1 Workstation. To control equal loading, blots were hybridized with a DIG-labeled probe against U6 snRNA.

For miR-153 in situ hybridization, mandibles were dissected from PN6 and PN9 BALB/c mice, and the surrounding soft tissues were partially retained in order to keep the integrity of the mandibles. After being fixed in 4% paraformaldehyde (PFA) at 4 °C overnight, the PN6 and PN9 hemimandibles were decalcified in 10% EDTA (pH 7.4) for 10 days and 14 days, respectively. Sagittal sections of 7 μm were prepared from paraffin embedded tissue blocks. Commercially available miRNA probes (Exiqon, Inc., MA, USA) were used to detect miR-153 expression in enamel organs overlying mandibular first molars and incisors. Details regarding the procedures of miRNA in situ hybridization were described in previous studies11 (link).