*3.5. RNA Isolation and Real-Time Reverse Transcript-Polymerase Chain Reaction (RT-PCR)*

RT-PCR was used to determine the gene expression of HIF-1α in HCT116 cells. Briefly, HCT116 cells (2 × 105 cells/mL) were cultured in 36-mm dishes for 24 h. The cells were then treated with various indicated concentrations of SA for an additional 8 h, with or without CoCl2. Total cellular RNA was extracted with TRIzol reagent according to the manufacturer's instructions. The total RNA (1 μg) that was isolated from the cells was used for reverse transcription reaction with Reverse Transcription Reagents. The cDNA was reverse transcribed at 42 ◦C for 60 min with 0.5 μg of oligo (dT)15 in a reaction volume of 20 μL using the reverse transcription system (Promega, MI, USA). Specific gene primers were designed and custom synthesized by Bioneer Corporation (Daejeon, Korea); HIF-1α forward: 5 -GATAGCAAGACTTTCCTCAGTCG-3 , reverse: 5 -TGGCCTCATATCCCATCAATTC-3 and GUSβ forward: 5 -CTACATCGATGATGACATCACCGTCAC-3 , reverse: 5 -TGCCCTTGACAGAGATCTGGTAA-3 . Real-time PCR was conducted using a MiniOpticon system (Bio-Rad, Hercules, CA, USA); each PCR amplification included 5 μL of reverse transcription product, iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA), and primers in a total volume of 20 μL. The following standard thermo cycler conditions were employed: 95 ◦C for 20 s prior to the first cycle; 40 cycles of 95 ◦C for 20 s, 56 ◦C for 20 s, and 72 ◦C for 30 s; 95 ◦C for 1 min; and 55 ◦C for 1 min. The threshold cycle (CT), indicating the fractional cycle number at which the amount of amplified target gene reached a fixed threshold for each well, was determined using the MJ Opticon Monitor software package (Bio-Rad, Hercules, CA, USA). Relative quantification, representing the change in gene expression in real-time quantitative PCR experiments between a sample-treated group and the untreated control group, was calculated by the comparative CT method in accordance with previously described methods [32]. The data were analyzed by evaluating the expression 2−ΔΔCT, where ΔΔCT = (CT of target gene − CT of housekeeping gene) treated group − (CT of target gene − CT of housekeeping gene) untreated control group. For the treated samples, the evaluation of 2−ΔΔCT represents the fold change in gene expression relative to the untreated control, normalized to a housekeeping gene (GUSβ).
