Superscript 3 platinum sybr green

For the extraction of total RNA from GBM cells and clinical tissues, we used TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) following the protocols of the manufacturer. We used SuperScript™ III Platinum™ SYBR™ Green (Thermo Fisher Scientific, Waltham, MA, United States) to generate the cDNA of mRNA and circRNA. We carried out quantitative real-time PCR (qRT-PCR) with SuperScript™ III Platinum™ SYBR™ Green (Thermo Fisher Scientific, Waltham MA, United States). GAPDH and U6 were used as internal controls. The TaqMan™ MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, United States) was utilized to obtain the cDNA of microRNA. The TaqMan™ MicroRNA Assay (Thermo Fisher Scientific, MA, United States) was used to carry out qRT-PCR. qRT-PCR was carried out utilizing ABI 7500 real-time PCR systems (Applied Biosystems, Foster City, CA, United States), and we calculated the RNA relative expression levels by using the 2-ΔΔCt method. The primers were obtained from Sangon Biotech (Shanghai, China). microRNA reverse primers were obtained from the TaqMan™ MicroRNA Assay (Thermo Fisher Scientific, Waltham, MA, United States). The primer sequences used for PCR amplification were as follows:

PDL tissues and HGF samples of each group were selected, and total RNA was extracted in three parallel copies. After determining the RNA concentration, cDNA was synthesized according to the instructions of the reverse transcription kit (18,064,071; SuperScript ™II, Thermo Fisher, Shanghai, China). Real-time quantitative RT-PCR was conducted according to the PCR kit instructions (11,746,500; SuperScript™ III Platinum™ SYBR™ Green; Invitrogen, Shanghai, China), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was selected as the reference gene to detect the mRNA expression of circ_0138959 and CASP5. U6 was used as an internal reference to detect the expression of miR-527 [21 (link)]. The primer sequences are shown in Table 1. The PCR procedure was conducted at 95°C for 120 s and 95°C for 20 s, 60°C for 25 s, and 72°C for 20 s, for 40 cycles. The data were analyzed using the 2^−ΔΔCt relative expression method. All experiments were repeated thrice.

Forty-eight cervical samples (14 normal, 15 CIN1-3, and 19 CCa) were obtained from the Seventh Affiliated Hospital affiliated to Sun Yat-sen University with the approval of the Hospital’s Protection of Human Subjects Committee. Informed consents were obtained from these donors. Total RNAs were isolated from cervical tissues with TRIzol reagent (Invitrogen, Carlsbad, CA). The cDNA was synthesized from 1 µg total RNA using M-MLV reverse transcriptase (Invitrogen) and oligo(dT)18 primers in accordance with the manufacturer’s protocol. qRT-PCR was carried out in triplicate with SuperScript™ III Platinum™ SYBR™ Green (Invitrogen) on a 7500 Real-Time PCR System (Thermo Fisher Scientific, MA, USA). The temperature protocol was 95°C for 20 min, followed by 40 cycles (95°C for 15 s, 59°C for 15 s). Beta-actin was used as an internal control. The relative expression of FNDC3B and BPGM was calculated using the 2^-ΔΔCT formula (19 (link)). The sequence-specific primer pair for FNDC3B was 5′-CAACAGCCCTCCTTCTTCTATCT-3′ (sense) and 5′-GCACCCTCTTTACTTCCAACTCAT-3′ (antisense). The sequence-specific primer pairs for BPGM was 5′-ATCAGAAACTCAACAGCGAAGG-3′ (sense) and 5′-TGTGAATGGACCGATTAAGGAC-3′ (antisense). The sequence-specific primer pair for beta-actin was 5′-CCACGAAACTACCTTCAACTCC-3′ (sense) and 5′-TCTTGATCTTCATTGTGCTGGG-3′ (antisense).