Opticon monitor analysis software v 2

Total RNA was extracted using TRIzol reagent (Life Technologies) according to the standard protocol. HOTAIR cDNA was obtained from total RNA using the Fast Quant RT Kit (TIANGEN). Real-time PCR was performed using Super Real Pre Mix Plus SYBR Green (TIANGEN) on an ABI-7500 Real-time PCR machine (Life Technologies).The following thermocycling protocol was employed: 95 °C for 30s, 60 °C for 30 s and 72 °C for 30 s for 40 cycles. GAPDH was used as the internal control. Data are presented as fold changes (2^−ΔΔCt) and were analyzed using the Opticon Monitor Analysis Software V 2.02 (MJ Research).

The total RNA was extracted from glioma cells using Trizol. RNA was reverse transcribed, and real‐time quantitative PCR was conducted on a Bio‐Rad Cycler system using the SYBER Premix DimerEraser. Fold changes were calculated by relative quantification. Data are shown as fold change (2‐ΔΔCT method) and analysed initially using Opticon Monitor Analysis Software V 2.02 software (MJ Research).

Quantitative Real-time PCR was used to confirm the circRNA expression profiles obtained from the microarray data. Total RNA was extracted from cells using Trizol Reagent (Invitrogen) and reversely transcribed into cDNA using Super Script TM III Reverse Transcriptase (Invitrogen) according to a standard protocol. The relative gene expression was determined using ViiA 7 Real-time PCR System (Applied Biosystems). All samples were normalized to the signal generated from GAPDH (Sangon Biotech, Shanghai, China). Data was shown as fold change (2−ΔΔCt) and analyzed initially using Opticon Monitor Analysis Software V2.02 (MJ Research, Waltham, MA, USA). Triplicates were performed for each sample in three independent experiments. Primer sequences were presented in Additional file 1: Table S1.