Bulge-Loop Real-Time RT-PCR

For quantitative evaluation of miRNAs, bulge-loop real-time RT-PCR was performed. The detailed procedure has been described in a previous study.11 In brief, the relative expression level of each miRNA was first normalized to small nuclear RNA U6 and then normalized to left group data values using 2^−DDCt methodology. All the bulge-loop RT primers were purchased from RiboBio (Guangzhou, China), and the real-time PCR analysis was performed according to the manufacturer’s instructions.11