Real-time quantitative PCR analysis of gene expression in human colon cancer cells treated with cottonseed-derived gossypol

Relative mRNA levels of 55 genes were evaluated with the SYBR Green qPCR assays after gossypol treatment for 8 h. Overall design: Human colon cancer cells (COLO 225) were purchased from ATCC. The cells in triplicate were treated with various concentrations of gossypol for 8 h. RNA isolation and cDNA synthesis were performed as described previously. Relative mRNA levels were evaluated with the SYBR Green qPCR assays using Bio-Rad reagents. BCL2 mRNA was selected as the internal reference for qPCR analysis for this cell type. 1% DMSO treatment was the sample control. The 2-ΔCT and 2-ΔΔCT method of relative quantification was used to determine the fold change in expression.