Validation of reference genes for normalization of real-time quantitative RT-PCR data in traumatic brain injury

Careful validation of reference genes used for the normalization of real‐time RT‐PCR data is required to obtain accurate results regarding gene expression. We evaluated the stability of seven commonly used reference genes in the cerebral cortex and hippocampus of rats 3 days following traumatic brai...

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Bibliographic Details
Published in:Journal of neuroscience research 2009-01, Vol.87 (1), p.34-41
Main Authors: Cook, Naomi L., Vink, Robert, Donkin, James J., van den Heuvel, Corinna
Format: Article
Language:English
Subjects:
Animals
Brain - metabolism
Brain - physiopathology
Brain Injuries - genetics
Brain Injuries - pathology
Disease Models, Animal
Gene Expression - physiology
Gene Expression Profiling - methods
Male
neurotrauma
Polymerase Chain Reaction - methods
Polymerase Chain Reaction - standards
quantitation
Rats
Rats, Sprague-Dawley
real-time PCR
Reference Standards
Reproducibility of Results
RNA - metabolism
substance P
Transcription Factors - genetics
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Summary:Careful validation of reference genes used for the normalization of real‐time RT‐PCR data is required to obtain accurate results regarding gene expression. We evaluated the stability of seven commonly used reference genes in the cerebral cortex and hippocampus of rats 3 days following traumatic brain injury (TBI). HPRT, SDHA, and GUSB were found to be the most stable reference genes in the cerebral cortex, whereas B2MG, TBP, and GAPDH were the most stable in the hippocampus. The use of three reference genes was determined to be the optimal number for accurate normalization of data. To illustrate this point, when our gene of interest, substance P (SP), was normalized against the three most stable reference genes in both brain areas, we found no significant difference between injured and uninjured rats at the 3‐day time point. However, when our SP data were normalized to each reference gene individually, SP mRNA level was highly variable depending on the reference gene chosen. The results of the present study highlight the importance of validating reference genes to be used for real‐time RT‐PCR analysis. The use of the most stable reference genes presented here will allow more accurate normalization of gene expression data in TBI. © 2008 Wiley‐Liss, Inc.
ISSN:0360-4012
1097-4547
DOI:10.1002/jnr.21846