Stepone

The RNA was extracted from fresh OS tissues, adjacent non-tumor tissues, OS cells and hFOB1.19 cells using the Qiagen RN easy FFPE protocol (Qiagen NV, Venlo, the Netherlands). The expression level of let-7a and AURKB was evaluated by qRT-PCR (StepOne^™; Bio-Rad Laboratories Inc., Hercules, CA, USA), and U6 snRNA and GAPDH were used as endogenous references. Total RNA in OS cells or tissues was extracted using TRIzol (Thermo Fisher Scientific). Reverse transcription was performed using Prime Script RT Reagent Kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer’s protocol. Then, each sample was analyzed by qPCR under the conditions described in the manufacturer’s instructions for SYBR Premix Ex Tap II (Thermo Fisher Scientific): 50°C for 2 minutes, 95°C for 2 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 30 seconds. Relative expression was calculated using the 2−ΔΔCt method. All of primer sequences are summarized in Table 1.