miRNA and Gene Expression Analysis

For first strand cDNA synthesis, 2 μg total RNA were extracted using PrimeScript™ RT reagent Kits accompanied with gDNA Eraser (TaKaRa, code no. RR047A) and miRNAs were extracted using miRcute Plus miRNA First-Strand cDNA Kits (TIANGEN, code no. KR211). miRNAs were quantified by stem-loop RT-PCR [32 (link)]. Gene expression was analyzed by qPCR using SYBR green supermixes from Bio-Rad and CFX96 real-time system. Each experiment was performed in three biological replicates. The expression of miRNAs and genes were calculated through the 2^{-ΔΔC (t)} method [53 (link)] with internal reference genes TBP and U6, respectively. Primers are listed in Additional file 11: Table S7. One-way ANOVA was used for statistical analyses in Microsoft Excel.