Analysis of intestinal adaptation gene expression by cDNA expression arrays

JPEN: Journal of Parenteral and Enteral Nutrition,  Nov/Dec 2000  by Erwin, Christopher R,  Falcone, Richard A Jr,  Stern, Lawrence E,  Kemp, Christopher J,  Warner, Brad W

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Another method to validate the results of the array is to compare them against previous reports of gene expression changes during intestinal adaptation. Ornithine decarboxylase,27 neu,28 glucose-regulated protein,29 and insulin-like growth factor binding protein-4(30) have been shown to be upregulated twofold or more after SBR. These results are consistent with the results obtained using the array system.

Two techniques were used to confirm the cDNA array results RT-PCR and Northern blot analysis. In Figure 3, the RT-PCR results for three genes that were upregulated in SBR relative to sham by array are shown. One of those genes, ornithine decarboxylase, has been shown to increase approximately twofold after SBR in rats.27 RT-PCR results correlated with those of the array, but with a difference magnitude. These differences existed despite the use of proprietary oligonucleotide primers for ornithine decarboxylase and other genes (data not shown) that amplify the exact cDNA sequence used on the array. This discrepancy also was observed with Northern blot analysis. Other investigators who have applied the Clontech mouse cDNA arrays to tissue culture systems have reported similar discrepances.25 In that study, only 75% of the positive results by array could be confirmed by Northern blot analysis with magnitude differences of 1.4 +/- 0.5-fold. In this study, considering both RT-- PCRs and Northern blots, 70% of the array results were confirmed with magnitude differences of 2.0 +/- 0.5-fold (similar number of genes tested in both studies). These magnitude differences may be attributable to characteristics inherent in the filter-based cDNA array technology and represent limitations of sensitivity for this method. Recent advances in cDNA array technology using fluorescently labeled cDNA probes and high-density oligonucleotides on a chip have greatly increased the sensitivity and accuracy.4,31

All of the genes used to confirm the array are related, in some manner, to the adaptation process. Glutathione reductase is involved in small intestine apoptosis.32 Members of the NF-2 family of transcription factors have a role in the induction of apoptosis.33 The oxidative stress-induced protein interacts with NF-2 family members,34 and oxidative stress promotes apoptosis in the small intestine.32 The etoposide-induced p53 responsive mRNA is a stress-response protein involved in p53-mediated apoptosis.36 Ornithine decarboxylase has previously been demonstrated to be upregulated approximately twofold after SBR.27 Basic Kruppel-like factor (BKLF), a transcription regulation factor, activates the promoter for insulin-like growth factor,36 which increases more than twofold in the ileum after SBR.30 Prothymosin-alpha is associated with accelerated cell proliferation,37 consistent with the increase in cell proliferation that occurs after SBR.7 None of these aforementioned genes, except ornithine decarboxylase, has previously been shown to be involved with intestinal adaptation.