Single color real time pcr system

Overall RNA was harvested from indicated cells utilizing TRIzol reagent (Invitrogen, USA). qRT-PCR was carried out on a Bio-Rad Single Color Real-Time PCR system (Bio-Rad Laboratories, Inc., USA) with SYBR-Green Real-Time PCR Master Mix (Toyobo Life Science, Japan). β-Actin was used as an internal control.

Total RNA was extracted from fresh tissues or cells by using TRIzol Reagent (Invitrogen, USA) in accordance with the manufacturer's instructions. The IDH1 expression of each sample was assessed using the Bio-Rad Single Color Real-Time PCR system (Bio-Rad, USA) with SYBR Green Real Time PCR Master Mix (TOYOBO, Osaka, Japan). The synthesized primer sequences (Sangon Biotech, China) were as follows: IDH1:5′-GTCGTCATGCTTATGGGGAT-3′ (forward primer), 5′-CAACACCACCACCTTCTTCA-3′ (reverse primer); GAPDH: 5′-GAAGGTCGGAGTCAACGGAT-3′ (forward primer), 5′-CCTGGAAGATGGTGATGGGAT-3′ (reverse primer). The IDH1 expression was calculated using the 2−ΔΔCt method, where ΔCt = Ct_IDH1–Ct_GADPH and ΔΔCt = ΔCt_test –ΔCt_control. All assays were performed in triplicate. The data are presented as the mean ± SD.

Surgical specimens were processed immediately after surgery. Total RNA was extracted from tissues using TRIzol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol. Complementary DNA (cDNA) was generated using qPCR RT Kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Primers were made by Sangon Biotech (Sangon Biotech, Shanghai, China). The primer sequences were as follows: Notch2 Forward primer: 5′-GGGACCCTGTCATACCCTCT-3′ and Reverse primer: 5′-GAGCCATGCTTACGCTTTCG-3′; β-actin Forward primer:5′-CAAAGGCCAACAGAGAGAAGAT-3′ and Reverse primer: 5′-TGAGACACACCATCACCAGAAT-3′. PCR was performed at 95 °C for 1 min and 40 cycles of 95 °C for 15 s, 58 °C for 15 s and 72 °C for 45 s. Notch2 expression was quantified using a Bio-Rad Single Color Real-Time PCR system (Bio-Rad, Hercules, California, USA) and calculated according to the mathematical model R = 2^−ΔΔCT, where ΔCT = CT_Notch2 − CT_β-actin, and ΔΔCT = ΔCT_test −ΔCT_control. All RT-PCRs were performed in triplicate, and the data are presented as the mean ± SD.