摘要
目的:研究转染质粒pcDNA3.1对内参基因表达影响差别,为今后在采用pcDNA3.1载体进行表达分析时内参基因的选择提供参考。方法:选用HepG2、HEK293、Hela、A549、QGY共5种细胞转染pcDNA3.1空载体质粒,转染后48h提取细胞总RNA荧光定量PCR法比较内参基因甘油醛-3-磷酸脱氢酶(Glyceraldehyde-3-phosphste dehydrogenase,GAPDH)、肌动蛋白(β-actin)、18sRNA表达水平差异,提取细胞总蛋白,检测GAPDH、β-actin表达水平差异。结果:5种细胞在转染质粒pcDNA3.1后,内参基因转录和蛋白表达均出现不同程度的下降,其中以β-actin下降最为明显,GAPDH及18sRNA在转录水平下降幅度小,且下降程度接近。结论:pcDNA3.1质粒转染造成基因表达非特异性抑制,其中对管家基因β-actin影响较大,因此在应用pcDNA3.1来源载体进行基因功能分析时,不推荐使用β-actin作为内参基因。
Objective：To investigate the effect of pcDNA3.1 vector on house keeping gene expression and set guide to further study work in using pcDNA3.1. Methods：5 cell lines were transfected with pcDNA3.1,48 h post transfection total RNA was extracted and applied for real time PCR to detect the RNA level of GAPDH、 β-actin、18sRNA difference.Meanwhile,extract total protein and analyze the GAPDH andβ-actin expression by Western blot.Results：RNA and protein levels all show of a decrease in 5 cell lines after transfection,with the most obviously β-actin.Conclusion：pcDNA3.1 might have non specifinhibition effect to cell,when using pcDNA3. 1 for gene function research,it is not recommend to use β- actin as reference gene.
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2010年第6期816-818,共3页
Journal of Chongqing Medical University
基金
国家自然科学基金(编号:30600520)
关键词
PCDNA3.1
内参基因
转染
转录
表达
pcDNA3.1
House keeping Gene
Transfection
Transcription
Expression
