Analysis of the effect of pcDNA3.1 on house keeping gene expression

CHEN Ke; et al

doi:10.13406/j.cnki.cyxb.2010.06.001

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Analysis of the effect of pcDNA3.1 on house keeping gene expression

更新时间：2024-01-17

- Analysis of the effect of pcDNA3.1 on house keeping gene expression
- Journal of Chongqing Medical University Vol. 35, Issue 6, Pages: 816-818(2010)
- 作者机构：
  
  重庆医科大学感染性疾病分子生物学教育部重点实验室
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.2010.06.001
  CLC： Q78
- Published：2010，
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[1]陈可,王增产.pcDNA3.1载体对内参基因表达影响分析[J].重庆医科大学学报,2010,35(06):816-818.

CHEN Ke, et al. Analysis of the effect of pcDNA3.1 on house keeping gene expression. [J]. Journal of Chongqing Medical University 35(6):816-818(2010)
[1]陈可,王增产.pcDNA3.1载体对内参基因表达影响分析[J].重庆医科大学学报,2010,35(06):816-818. DOI： 10.13406/j.cnki.cyxb.2010.06.001.

CHEN Ke, et al. Analysis of the effect of pcDNA3.1 on house keeping gene expression. [J]. Journal of Chongqing Medical University 35(6):816-818(2010) DOI： 10.13406/j.cnki.cyxb.2010.06.001.

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。

Abstract

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.

关键词

pcDNA3.1内参基因转染转录表达

Keywords

pcDNA3.1House keeping GenetransfectionTranscriptionexpression

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Related Institution

Department of Pediatric Research Institute,the Children’s Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing Key Laboratory of Pediatrics,Chongqing International Science and Technology Cooperation Center for Child Development and Disorders

浙江医科大学传染病研究所

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Department of Surgery,College of Pediatrics,Chongqing Medical University

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