Transcriptional levels of miRNAs were monitored using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Total RNA was extracted using TRIzol total RNA isolation reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. The quality of RNA was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) [35 (link)]. Total RNA (1 μg) was used for cDNA synthesis with the First-Strand cDNA Synthesis Kit (Fermentas, Burlington, Canada). Real-time PCR was performed as follows: 95°C for 15 min for hot-start, followed by 95°C for 30 s, and 60°C for 60 s for 40 cycles, using the SYBR Green Quantitative PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on a Bio-Rad IQ5 instrument (Bio-Rad, Hercules, CA, USA). Primers for amplification of microRNA (RiboBio, Guangzhou, China) are listed in Table 1. Each reaction was performed in triplicate and measured using the comparative Ct (2−ΔΔCt) method with U6 snRNA as the normalization control.