Stem-loop qRT-PCR for miRNA Expression

We used stem-loop qRT-PCR to confirm the miRNA expression levels^{71 (link)}. For selected miRNAs, ~1 μg DNA-free total RNA was hybridised with miRNA-specific stem-loop RT primers. The hybridised molecules were reverse transcribed into cDNAs using a Superscript III kit (Thermo Fisher Scientific). We designed forward miRNA-specific primers for the mature miRNA sequences and used a universal reverse primer for the stem-loop sequences. Reactions were repeated three times for each sample set. Each 20-μL reaction mixture contained 1 μL cDNA, 10 μL 2 × FastStart SYBR Green (Roche) and 0.8 μL forward and reverse primers (TaKaRa, Ohtsu, Japan). The PCR amplification conditions were as follows: 95 °C for 10 s and 60 °C for 30 s. The PCRs were conducted using the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Data were analysed using the 2^−ΔΔCt method to calculate relative gene expression^{72 (link)}. Supplementary Table S8 lists all primers used in the qRT-PCR experiments, including those for miRNAs and their targets.