Evaluating Gene Expression in HT-29 Cells

Given that, following the cell viability test, the most affected cell line was HT-29, it was decided that the influence of 5FU, PBT, and PBT-5FU on gene expression should be established by applying the RT-PCR method [15 (link)] to this cell line. To evaluate the expression of the Bax, Bcl-2 (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and Bad (Eurogentec, Seraing, Belgium), the cells (1,000,000 cells/well) were cultured in 6-well plates. After reaching a confluence of approximately 80%, the cells were stimulated with test samples for a period of 72 h. After this time, RNA was isolated using Trizol reagent and the Quick-RNA™ purification kit, and its amount was determined using a DS-11 spectrophotometer (DeNovix, Wilmington, DE, USA). Finally, RNA transcription was completed using the Maxima^® First Strand cDNA Synthesis Kit, and quantitative real-time PCR analysis was performed using the Quant Studio 5 real-time PCR system (Thermo Fisher Scientific, Inc., Waltham, MA, USA) in the presence of Power SYBR-Green PCR Master Mix.