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

本研究采用SYBR Green实时定量聚合酶链式反应（SYBR Green qPCR），检测经棉酚处理8小时后的55个基因的相对mRNA表达水平。实验总体设计如下：人结肠癌细胞COLO 225购自美国典型培养物保藏中心（ATCC）。将细胞设置三个生物学重复，分别以不同浓度的棉酚处理8小时，以1%二甲基亚砜（DMSO）处理组作为样本对照。按照既往报道的方法完成RNA提取与互补脱氧核糖核酸（cDNA）合成。采用伯乐（Bio-Rad）试剂，通过SYBR Green qPCR检测相对mRNA表达水平，本次实验选取BCL2 mRNA作为该细胞系的qPCR内参基因。采用2-ΔCT法与2-ΔΔCT法进行相对定量分析，以计算基因表达的倍数变化。