Reverse transcription quantitative-PCR (RT-qPCR) analysis
Stem-loop RT-PCR was used to quantify miRNAs and target genes according to a previous study (Liu et al., 2014). Briefly, 3 μg RNA was reverse-transcribed to cDNA by using stem-loop primers and SuperScript III RT (Invitrogen). The reaction procedure was the following steps: 16°C for 30 min; 60 cycles of 30°C for 30 s, 42°C for 30 s; and then 70°C for 5 min to inactivate the reverse transcriptase.
RT-qPCR was performed using the ABI Prism 7500 system (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s protocol. The thermocycling program was as follows: 95°C for 1 min; 40 cycles of 95°C for 10 s, 60°C for 40 s.