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. 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.

采用SYBR Green定量实时聚合酶链反应（SYBR Green qPCR），检测经棉酚（gossypol，缩写Gos）处理2~24小时后的27个基因的相对mRNA水平。本研究所用的小鼠巨噬细胞RAW264.7细胞（TIB-71），源自经Abelson鼠白血病病毒（ATCC）诱导的雄性小鼠肿瘤组织。将细胞以三重平行重复的方式培养，分别用不同浓度的棉酚处理2、8和24小时。RNA提取与cDNA合成操作参照既往已发表的实验方法进行。采用伯乐（Bio-Rad）公司的试剂，通过SYBR Green qPCR检测相对mRNA水平。针对该细胞系，选取Rpl32 mRNA作为qPCR分析的内参基因。以1%二甲基亚砜（DMSO）处理作为样本对照组。采用2^(-ΔCT)与2^(-ΔΔCT)相对定量法计算基因表达的倍数变化。