Applied biosystems quantstudio 1 real time pcr system

Total RNA was extracted using easy-BLUE (iNtRON Biotechnology, Dajeon, Korea) in accordance with the manufacturer’s protocol. cDNA was synthesized from 2 µg of total RNA using TOPscript^TM Drymix Kit (Enzynomics, Dajeon, Korea). qRT-PCR was performed using TB Green ^® Premix Ex Taq^TM II (Tli RNAse H Plus) (TaKaRa, Japan) with Applied Biosystems QuantStudio™ 1 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Gene expression data were normalized based on the use of the same amount of total RNA samples. The relative fold change of gene expression was calculated using the ∆Ct method. Sequences of primers are listed in Table 2.

The human A549 lung cancer cells were cultured in RPMI-1640 replenished with 10% fetal bovine serum (FBS). All cells were cultured at 37°C in a humidified 5% CO₂ incubator. The digested cells were counted and inoculated in six-well plates until cell attachment, and then, cells were cultured in a medium added with 100 ng/ml recombinant TNFSF12 (R&D Systems) or 200 ng/ml recombinant SPP1 (R&D Systems), respectively. After 48-h culture, the total RNA was isolated from cells using TRIzol reagent (Magen) according to the manufacturer’s protocol. Reverse-transcribed complementary DNA was synthesized using the Evo-M-MLV RT Kit (AG11705, Accurate Biotechnology). qRT-PCR was performed using the Applied Biosystems QuantStudio 1 Real-Time PCR System (Thermo Fisher) and the PowerUp^TM SYBR Green Mix (Thermo Fisher). The fold-change in the expression of target genes was calculated by the 2^–ΔΔCt method. The primer sequence is listed in Supplementary Table 4.