Validation of endogenous internal real-time PCR controls in renal tissues.
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 RT-PCR analyses and Affymetrix GeneChip 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.


CONCLUSIONS
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.
Cui
Xiangqin
X
Department of Medicine, University of Alabama at Birmingham, 1900 University Blvd, Tinsley Harrison Tower 611J, Birmingham, AL 35294, USA.
Zhou
Juling
J
Qiu
Jing
J
Johnson
Martin R
MR
Mrug
Michal
M
D000818:Animals; D021984:Cyclophilin A / Q000235:genetics; D020869:Gene Expression Profiling / Q000592:standards*; D023281:Genomics / Q000592:standards; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D007668:Kidney / Q000502:physiology*; D052177:Kidney Diseases, Cystic / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D020411:Oligonucleotide Array Sequence Analysis / Q000592:standards*; D010735:Phosphoglycerate Kinase / Q000235:genetics; D011786:Quality Control; D012016:Reference Values; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*
D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C528165:Pgk1 protein, mouse; D010735:Phosphoglycerate Kinase; D021984:Cyclophilin A