RNA Extraction and miRNA Quantification

Total RNA was extracted from fresh-frozen tissues or cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and miRNA molecules were purified using the mirVana miRNA isolation kit (Ambion, Austin, USA) as previously described [38 (link)]. RNA was reverse transcribed using the PrimeScript RT reagent kit (Takara, Dalian, China). qRT-PCR was performed using SYBR Premix Ex Taq II (Takara) on an ABI Prism 7500 PCR system (Applied Biosystems, USA). Data were normalized to β-actin. Mature miR-148a was analyzed by qRT-PCR using the TaqMan MicroRNA Assay Kit (Applied Biosystems). The expression of mature miR-148a was determined by real-time PCR analysis following stem-loop RT and data were normalized to U6 snRNA. Relative expression was measured using the 2^−ΔΔCT method [39 (link)] with the following primers: GADD45A forward: 5′- GAGCAGAAGACCGAAAGCGAC-3′, reverse: 5′-GAATGTGGATTCGTCACCAGC-3′. MiR-148a RT-primer: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACAAAGTT-3′; MiR-148a PCR forward: 5′-GCTAGTGTTCTGAGACACTCCG-3′, PCR reverse: 5′-GTGCAGGGTCCGAGGT-3′. U6 RT-primer: 5′-CGCTTCACGAATTTGCGTGTCAT-3′, U6 PCR forward: 5′-GCTTCGGCAGCACATATACTAAAAT-3′, PCR reverse: 5′-CGCTTCACGAATTTGCGTGTCAT-3′.