Approach for defining endogenous reference genes in gene expression experiments

被引:85
作者
García-Vallejo, JJ
Van het Hof, B
Robben, J
Van Wijk, JAE
Van Die, I
Joziasse, DH
Van Dijk, W [1 ]
机构
[1] Free Univ Amsterdam, Med Ctr, Dept Mol Cell Biol & Immunol, NL-1081 BT Amsterdam, Netherlands
[2] Free Univ Amsterdam, Med Ctr, Dept Pediat, NL-1081 BT Amsterdam, Netherlands
关键词
endogenous reference gene; housekeeping gene; gene expression analysis; real-time PCR; HUVEC; endothelial cells; B cells; IgA nephropathy;
D O I
10.1016/j.ab.2004.02.037
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The quantification of gene expression by real-time polymerase chain reaction (PCR) has revolutionized the field of gene expression analysis. Due to its sensitivity and flexibility it is becoming the method of choice for many investigators. However, good normalization protocols still have to be implemented to facilitate data exchange and comparison. We have designed primers for 10 unrelated genes and developed a simple protocol to detect genes with stable expression that are suitable for use as endogenous reference genes for further use in the normalization of gene expression data obtained by real-time PCR. Using this protocol, we were able to identify human proteosome subunit Y as a reliable endogenous reference gene for human umbilical vein endothelial cells treated for up to 18 h with TNFalpha, IL-4, or IFNgamma and for B cells isolated from healthy controls and patients suffering from IgA nephropathy. Other optional endogenous reference genes that can be considered are phosphomannomutase (PPMM) and actin for endothelial cells and glyceraldehyde-3-phosphate dehydrogenase and PPMM for B cells. (C) 2004 Elsevier Inc. All rights reserved.
引用
收藏
页码:293 / 299
页数:7
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