Sybr green master mix system

Total RNA was obtained from NCI-H520, A549, H358 and MRC-5 cells prior or the tumor tissues with Chanti-MACC-1 by using RNAeasy Mini kit (24 (link)) (Qiagen Sciences, Inc., Gaithersburg, MD, USA). A total of 1 µg total RNA was then transcribed into cDNA using the PrimeScript™ RT Master mix (Perfect Real Time; Takara Biotechnology Co., Ltd.) in an ABI PRISM 7900 real time system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The quality of the synthetic cDNA was verified by electrophoresis. Subsequently, the synthetic cDNA (10 ng) was subjected to RT-qPCR using SYBR-Green Master Mix system (Bio-Rad Laboratories, Inc). The protocol of thermos cycling was as follows: Denaturation, 95°C for 2 min; annealing, 40 repetitions of 95°C for 30 sec and 60°C for 60 sec; and final extension, 72°C for 10 min. The primers used in the present study were synthesized by Shanghai Shenggong Biology Engineering Technology Service, Ltd., Shanghai, China. MACC-1 forward, 5′-AGTGGGATTGTGGAGACGGTGT-3′ and reverse, 5′-AGGTAAAAGGAACTGGCAACGC-3′; GAPDH forward, 5′-GTGGACATCCGCAAAGAC-3′ and reverse, 5′-AAAGGGTGTAACGCAACTA-3′. GAPDH was included as an internal control. Differences in mRNA expression alterations were calculated by 2^−ΔΔCq (25 (link)). The results are expressed as the n-fold way compared with control.

Total RNA was extracted from H358 cells (1×10⁷) following treatment with Calotropin (0.50 mg/ml) for 48 h at 37°C using the RNAeasy Mini kit (Qiagen, Inc., Valencia, CA, USA). Total RNA (1 µg) was transcribed into cDNA at 37°C for 2 h using the QuantiNova Reverse Transcription kit (Qiagen, Inc.) and the quality was confirmed by electrophoresis. The cDNA (10 ng) was subjected to RT-qPCR using the SYBR Green Master Mix system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). PCR amplification was preliminary denaturation at 95°C for 60 sec, followed by 45 cycles of 95°C for 30 sec, annealing at 58°C for 30 sec, and 72°C for 30 sec in a total volume of 20 µl containing 50 ng of genomic DNA, 200 µM dNTP, 2.5 units of Taq DNA polymerase, and 200 µM of each primer. All of the forward and reverse primers were synthesized by Invitrogen (Table I; Thermo Fisher Scientific, Inc.). Relative mRNA expression was calculated using the 2^−ΔΔCq method (15 (link)) and the results are expressed as the n-fold way compared with the control.

Total RNA was extracted from A549 cells (1×10⁸) pre- or post-treatment with AbMTA2 (5 mg/ml, 1×10⁸), siMTA2 transfection (1×10⁸) or pMTA2 transfection (1×10⁸) using an RNAeasy Mini kit (Qiagen Sciences, Inc., Gaithersburg, MD, USA) according to the manufacturer's instructions. Total RNA (1 µg) was reverse transcribed into cDNA using the Reverse Transcription kit (Qiagen Sciences, Inc.) and the quality was confirmed using 1.5% agarose (Sigma-Aldrich; Merck KGaA) electrophoresis. cDNA (10 ng) was subjected to qPCR analysis with SYBR Green Master Mix system (Bio-Rad Laboratories, Inc.), according to the manufacturer's instructions. All the forward and reverse primers were synthesized by Invitrogen; Thermo Fisher Scientific, Inc. (Table I). Cycling conditions were as follows: 45 cycles of denaturation at 95°C for 2 min, annealing at 66°C for 30 sec with touchdown to 56°C for 30 sec, and extension at 72°C for 10 min. Relative mRNA expression changes were calculated using the 2^−ΔΔCq method (42 (link)). Experiments were repeated three times and the results are expressed as the n-fold change relative to the control.