Normalizing genes for quantitative RT-PCR in differentiating human intestinal epithelial cells and adenocarcinomas of the colon

被引:144
作者
Dydensborg, AB [1 ]
Herring, E [1 ]
Auclair, J [1 ]
Tremblay, E [1 ]
Beaulieu, JF [1 ]
机构
[1] Univ Sherbrooke, Fac Med, Dept Anat & Biol Cellulaire, Canadian Inst Hlth Res Grp Funct Dev & Physiopath, Sherbrooke, PQ J1H 5N4, Canada
来源
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY | 2006年 / 290卷 / 05期
关键词
mRNA analysis; intestinal cell differentiation; colon cancer cells;
D O I
10.1152/ajpgi.00234.2005
中图分类号
R57 [消化系及腹部疾病];
学科分类号
摘要
As for other mRNA measurement methods, quantitative RT-PCR results need to be normalized relative to stably expressed genes. Widely used normalizing genes include beta-actin and glyceraldehyde-3-phosphate dehydrogenase. It has, however, become clear that these and other normalizing genes can display modulated patterns of expression across tissue types and during complex cellular processes such as cell differentiation and cancer progression. Our objective was to set the basis for identifying normalizing genes that displayed stable expression during enterocytic differentiation and between healthy tissue and adenocarcinomas of the human colon. We thus identified novel potential normalizing genes using previously generated cDNA microarray data and examined the alterations of expression of two of these genes as well as seven commonly used normalizing genes during the enterocytic differentiation process and between matched pairs of resection margins and primary carcinomas of the human colon using real-time RT-PCR. We found that ribosomal phosphoprotein P0 was particularly stable in all intestinal epithelial cell extracts, thereby representing a particularly robust housekeeping reference gene for the assessment of gene expression during the human enterocytic differentiation process. On the other hand, beta-2-microglobulin generated the best score as a normalizing gene for comparing human colon primary carcinomas with their corresponding normal mucosa of the resection margin, although others were found to represent acceptable alternatives. In conclusion, we identified and characterized specific normalizing genes that should significantly improve quantitative mRNA studies related to both the differentiation process of the human intestinal epithelium and adenocarcinomas of the human colon. This approach should also be useful to validate normalizing genes in other intestinal contexts.
引用
收藏
页码:G1067 / G1074
页数:8
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