Stem-loop reverse transcription and real-time polymerase chain reaction

A total of 1 μg of total RNA was reverse-transcribed in a stem-loop reverse transcription (RT) reaction by using RT primers and SuperScript III Reverse Transcriptase according to the user’s manual (Invitrogen, Carlsbad, CA, USA). The reaction was performed under the following incubation conditions: 30 min at 16 °C, followed by 50 cycles of 20 °C for 30 s, 42 °C for 30 s, and 50 °C for 1 s. The enzyme was subsequently inactivated through incubation at 85 °C for 5 min. Real-time polymerase chain reaction (PCR) was performed using a miRNA-specific forward primer and a universal reverse primer and involved incubation at 94 °C for 10 min, followed by 40 cycles of 94 °C for 15 s and 60 °C for 32 s. The gene expression level was detected using the SYBR Green I assay (Applied Biosystems, Foster City, CA, USA), and the expression levels of miRNAs were normalised to those of U6. The primer sequences used to examine miRNAs are listed in Supplementary Table S2.