Journal
AMERICAN JOURNAL OF NEPHROLOGY
Volume 30, Issue 5, Pages 413-417Publisher
KARGER
DOI: 10.1159/000235993
Keywords
Real-time PCR; Kidney disease; Endogenous internal control; Reference gene; Housekeeping gene
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Funding
- Polycystic Kidney Disease Foundation
- UAB-UCSD O'Brien Center [1P30 DK079337]
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [P30DK079337] Funding Source: NIH RePORTER
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Background: Endogenous internal controls ('reference' or 'housekeeping' genes) are widely used in real-time PCR (RT-PCR) analyses. Their use relies on the premise of consistently stable expression across studied experimental conditions. Unfortunately, none of these controls fulfills this premise across a wide range of experimental conditions; consequently, none of them can be recommended for universal use. Methods: To determine which endogenous RT-PCR controls are suitable for analyses of renal tissues altered by kidney disease, we studied the expression of 16 commonly used 'reference genes' in 7 mildly and 7 severely affected whole kidney tissues from a well-characterized cystic kidney disease model. Expression levels of these 16 genes, determined by TaqMan (R) RT-PCR analyses and Affymetrix GeneChip (R) arrays, were normalized and tested for overall variance and equivalence of the means. Results: Both statistical approaches and both TaqMan- and GeneChip-based methods converged on 3 out of the 4 top-ranked genes (Ppia, Gapdh and Pgk1) that had the most constant expression levels across the studied phenotypes. Conclusion: A combination of the top-ranked genes will provide a suitable endogenous internal control for similar studies of kidney tissues across a wide range of disease severity. Copyright (C) 2009 S. Karger AG, Basel
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