qRT-PCR analysis
Total RNA from the samples was extracted using TRIzol (Takara, Dalian, China) followed by an RNasefree treatment (Takara). The RNA (4 μg) was pretreated with RQ1 Dnase I (Promega, Madison, WI, USA) to remove contaminating genomic DNA. Total RNA concentrations were measured using a NanoDrop instrument (Thermo Fisher Scientific, Waltham, MA, USA), and RNA quality was checked using nondenaturing agarose gel electrophoresis. Total DNA-free RNA (2 μg) was used for cDNA synthesis in a total volume of 40 μL, and the resulting cDNA was used as the template for RT-PCR. PCR for the target mRNAs was carried out in 20-μL reactions containing 2 μL cDNA synthesis reaction mixture, 400 nM each primer, and 10.5 μL SYBR Green PCR Master Mix (TianGen Biotech, Beijing, China) on an ABI 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA). The main feature of mature miRNA expression detection is the RT primer, which has a stem loop structure and a consensus sequence that effectively binds to the 3’end of the miRNA. PCR for the mature miRNAs was carried out in 25-μL reactions containing 2.5 μL cDNA synthesis reaction mixture, 400 nM each primer, and 12.5 μL SYBR Green PCR Master Mix. In addition, each measurement was repeated using three technical replicates, in which the RNA samples were mixed with three biological replicates. The gene primers are listed in Supplementary Table S1.