Comprehensive RNA Analysis Protocol

Total RNA was isolated using the Direct-zol™ RNA kit (Zymo Research). Before isolation, calvaria (2-to-3-day old mice) and other tissues (eight-week-old mice) were homogenized for 30 s in ice-cold QIAzol using the Ultra Turrax T25 tissue homogenizer (IKA). RNA was quantified using the NanoDrop 2000 spectrophotometer (Thermo Fischer Scientific).
For mRNA analysis, RNA was reverse transcribed into cDNA by the SuperScript III First-Strand Synthesis System (Invitrogen). Gene levels were assessed using real-time PCR using the QuantiTect SYBR Green PCR Kit (Qiagen) and the CFX384 Real-Time System machine (BioRad). Transcript levels were quantified using the 2^ΔΔCt method and normalized to the housekeeping gene Gapdh (set at 100). Primer pairs are shown in Suppl Table 3.
For miRNA quantification, miRNAs were reverse transcribed into cDNA using the Mir-X miRNA First-Strand Synthesis Kit as instructed (Takara). Similar to mRNA, miRNA levels were determined using QuantiTect SYBR Green PCR Kit and the CFX384 Real-Time System machine. miRNA quantification was assessed using a universal reverse primer provided in First-Strand Synthesis Kit (Takara) and miRNA specific forward primers listed in Suppl Table 3. As with mRNA, miRNA levels were quantified using the 2^ΔΔCt method and normalized to miR-103a-3p (set at 100)^{71 (link)}.