*2.3. Exploration and Validation of CRC-Related Genes Based on Real-Time PCR and Western Blotting*

To further validation of the RNA sequencing data generated by the above-mentioned parameters, the differential expression of top five genes including two upregulated (GADD45G and ATF3) and three downregulated genes (BUB1B, CCNA2 and CDK1) were evaluated on all treated and non-treated samples in both mRNA and protein levels. Total RNAs were firstly converted to cDNA using an c (AddBio Co., Seoul, Korea). According to the manufacturer's instructions, quantitative PCR analysis was performed by employing Add SYBER Master kit (AddBio Co., Seoul, Korea) on the CFX96 thermal cycler (Bio-Rad Co., Hercules, CA, USA). All the primers (Table 1) were designed by Gene Runner version 6 (http://generunner.net, 1 September 2020), and β-actin was used as internal control. The quantification of expression levels was studied by the 2−ΔΔCt method. Furthermore, melting curves were run to confirm the specificity and consistency of the products.


**Table 1.** Details of primers used in real-time PCR. F: forward; R: reverse.

Total protein was extracted from all samples using 500 μL of lysis buffer (Tris-HCl pH 8, 0.08 g NaCl, 0.003 g EDTA, 0.025 g sodium deoxycholate, 0.01 g SDS, and 1% NP40 enriched with an anti-protease cocktail). Thereafter, 10 μg of protein was electrophoresed using 10% SDS-PAGE at 120 V for 45 min and then transferred onto polyvinylidene difluoride membranes at 120 V for 1.5 h. The membranes were incubated with appropriate primary antibodies (all from Santa Cruz Biotechnology, Dallas, TX, USA) at 4 ◦C overnight. After

three-time PBS wash, the membranes were incubated with appropriate HRP-conjugated secondary antibodies (Cat no: sc-516102 and sc-2357; Santa Cruz Biotechnology, USA) for 1 h at room temperature. The immunoblots were detected on X-ray films using chemiluminescence ECL solution (Bio-Rad, Herecules, CA, USA). β-actin (Cat no: sc-47778; Santa Cruz Biotechnology, Inc.) was considered as internal control for normalization.
