Real-time quantitative PCR analysis of gene expression in mouse macrophages treated with cottonseed-derived gossypol

Relative mRNA levels of 27 genes were evaluated with the SYBR Green qPCR assays after gossypol (Gos) treatment for 2-24 h. Overall design: Mouse macrophages (RAW264.7 cells, TIB-71) was established from a tumor in a male mouse induced with the Abelson murine leukemia virus (ATCC). The cells in triplicate were treated with various concentrations of gossypol for 2, 8 and 24 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. Rpl32 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.