Primescipt rt kit

Total RNAs were extracted using the miRNeasy Mini Kit (Qiagen, Venlo, The Netherlands). RNA was reversely transcribed using PrimeScipt RT Kit (Takara). miR-431 was reversely transcribed using specific tailing reaction kit (B532451, Sangon, Shanghai, China). RT-qPCR was carried out on the 7500 Sequence Detection System (ABI, Foster City, CA) using SYBR Premix Ex Taq (Takara). The 2^-ΔΔCt method was used to quantify relative expression levels of target genes. For circ_0072464, total RNAs were incubated with or without 3 U/μg of RNase R (Epicentre, San Diego, CA) at 37°C for 20 min, and the resultant RNA was subsequently purified using the RNeasy MinElute Cleanup Kit (Qiagen). The circRNA was amplified using the specific divergent primers for the back-splice junction of circ_0072464. The agarose gel electrophoresis and sequencing were conducted to detect the amplified products. U6 and β-actin served as internal reference for miRNA and mRNA, respectively. All primers are shown in Table S1.

Total RNA was isolated from cell lines and xenograft tissue with a TRIzol (Ambion) or RNaesy Kit (Qiagen), following the manufacturer’s protocol. RNA was DNase-digested using a TURBO-DNA-free kit (Ambion). Reverse transcription of RNA was conducted using a RevertAid RT Kit (Thermo Fisher scientific) or PrimeScipt RT Kit (TaKaRa, Beijing, China). Analysis of cDNA was performed using either GoTaq qPCR Master Mix (Promega) as described by the manufacturer using a CFX Connect Real-Time System (Bio-Rad), StepOne Real-Time PCR System (Applied Biosystems), or standard PCR. Primers are listed in Table 2. Each (RT)-qPCR reaction was performed in at least three independent biological replicates. Data are shown as means (±s.d.). For indicated experiments, a two-sided t-test was used to assess statistical significance. The ∆Ct value was determined by subtracting the Ct value of housekeeper Ct value (GAPDH, β-actin) from the target Ct value (AURKA, Survivin/BIRC-5, MYBL2, CCNB1, CCNB2, CCNE1, CDC25C, E2F1, FOXM1, HISTH1B, KIF23, MKI67, p21^CIP1/CDKN1A, AR, TAPIR-1, and TAPIR-2).