Quantitative real‑time polymerase chain reaction
KYSE150 cells were treated with DMSO (0.1%) or ixazomib (10 μM) for
24h. Total RNA was isolated using the Trizol reagent and Ultrapure RNA
kit (CW Biotech, China) according to the manufacturer’s instructions. 2
mg of total mRNA was reverse-transcribed with the Superscript™ reverse
transcription system (Takara, Dalian, Japan). Quantitative real-time
reverse transcription polymerase chain reaction (RT-PCR) reaction was
carried out on ABI 7500 Real-Time PCR system (Applied Biosystems, Foster
City, CA, USA) using SYBR Green PCR master mix reagents (Takara, Dalian,
Japan). Relative quantification of target gene mRNA level was calculated
after normalization to GAPDH mRNA using the 2-△△Ct method. The primer
sequences were selected based on the published literature
(Du et al. , 2017).