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Research Article
Validation of reference genes for normalization of real-time quantitative RT-PCR data in traumatic brain injury
Article first published online: 18 AUG 2008
DOI: 10.1002/jnr.21846
Copyright © 2008 Wiley-Liss, Inc.
Additional Information
How to Cite
Cook, N. L., Vink, R., Donkin, J. J. and van den Heuvel, C. (2009), Validation of reference genes for normalization of real-time quantitative RT-PCR data in traumatic brain injury. Journal of Neuroscience Research, 87: 34–41. doi: 10.1002/jnr.21846
Publication History
- Issue published online: 16 DEC 2008
- Article first published online: 18 AUG 2008
- Manuscript Accepted: 12 JUN 2008
- Manuscript Revised: 12 MAY 2008
- Manuscript Received: 10 FEB 2008
Funded by
- Neurosurgical Research Foundation of Australia
- NHMRC Dora Lush Biomedical Postgraduate Scholarship
- Abstract
- Article
- References
- Cited By
Keywords:
- real-time PCR;
- substance P;
- quantitation;
- rats;
- neurotrauma
Abstract
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.