HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: 30/3/363    most recent 00251.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Kidd, M. Articles by Modlin, I. Search for Related Content PubMed PubMed Citation Articles by Kidd, M. Articles by Modlin, I.

GeneChip, geNorm, and gastrointestinal tumors: novel reference genes for real-time PCR

¹ Gastrointestinal Research Group, Yale University School of Medicine, New Haven, Connecticut
³ Keck Laboratory, Yale University School of Medicine, New Haven, Connecticut
² Department of Computer Science and Applied Mathematics, Weizmann Institute of Science, Rehovot, Israel
⁴ Institute of Pathophysiology, Centre for Molecular Medicine, Medical University of Graz, Austria

Accurate quantitation of target genes depends on correct normalization. Use of genes with variable tissue transcription (GAPDH) is problematic, particularly in clinical samples, which are derived from different tissue sources. Using a large-scale gene database (Affymetrix U133A) data set of 36 gastrointestinal (GI) tumors and normal tissues, we identified 8 candidate reference genes and established expression levels by real-time RT-PCR in an independent data set (n = 42). A geometric averaging method (geNorm) identified ALG9, TFCP2, and ZNF410 as the most robustly expressed control genes. Examination of raw C_T values demonstrated that these genes were tightly correlated between themselves (R² > 0.86, P < 0.0001), with low variability [coefficient of variation (CV) <12.7%] and high interassay reproducibility (r = 0.93, P = 0.001). In comparison, the alternative control gene, GAPDH, exhibited the highest variability (CV = 18.1%), was significantly differently expressed between tissue types (P = 0.05), was poorly correlated with the three reference genes (R² < 0.4), and was considered the least stable gene. To illustrate the importance of correct normalization, the target gene, MTA1, was significantly overexpressed (P = 0.0006) in primary GI neuroendocrine tumor (NET) samples (vs. normal GI samples) when normalized by geNorm_ATZ but not when normalized using GAPDH. The geNorm_ATZ approach was, in addition, applicable to adenocarcinomas; MTA1 was overexpressed (P < 0.04) in malignant colon, pancreas, and breast tumors compared with normal tissues. We provide a robust basis for the establishment of a reference gene set using GeneChip data and provide evidence for the utility of normalizing a malignancy-associated gene (MTA1) using novel reference genes and the geNorm approach in GI NETs as well as in adenocarcinomas and breast tumors.

GAPDH; housekeeping; microarray