Abstract
Accurate normalization is the prerequisite for obtaining reliable results in the quantification of gene expression. Using TaqMan Real Time RT-PCR, we carried out an extensive evaluation of five most commonly used endogenous controls, gapdh, β-actin, 18S rRNA, hprt and cypB, for their presumed stability of expression, in rat cortex and hippocampus, during aging, under dietary restriction and dexamethasone treatment. Valid reference genes (HKGs) were identified using GeNorm and Norm-Finder software packages and by direct comparison of Ct values. Analysis revealed gapdh and β-actin as the most stable HKGs for all treatments analyzed, combined or separately, in the cortex, while in the hippocampus gapdh/hprt and β-actin/hprt are the combination of choice for the single or combined effects of dietary restriction/dexamethasone, respectively. All treatments significantly influenced expression of 18S rRNA and cypB in both structures. In addition, we used gapdh and normalization factor, calculated by GeNorm, to compare the expression of α-syn in the cortex. Our results demonstrate the importance of the right choice of HKG and suggest the appropriate endogenous control to be used for TaqMan RT-PCR analysis of mRNA expression in rat cortex and hippocampus for selected experimental paradigms.
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Agardh, E., Gustavsson, C., Hagert, P., Nilsson, M., & Agardh, C. D. (2006). Modifying a standard method allows simultaneous extraction of RNA and protein, enabling detection of enzymes in the rat retina with low expressions and protein levels. Metabolism, 55, 168–174.
Andersen, C. L., Jensen, J. L., & Ørntoft, T. F. (2004). Normalization of real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Research, 64, 5245–5250.
Barrachina, M., Castano, E., Dalfo, E., Maes, T., Buesa, C., & Ferrer, I. (2006). Reduced ubiquitin C-terminal hydrolase-1 expression levels in dementia with Lewy bodies. Neurobiology of Disease, 22, 265–273.
Bhatia, P., Taylor, W. R., Greenberg, A. H., & Wright, J. A. (1994). Comparison of glyceraldehyde-3-phosphate dehydrogenase and 28S-ribosomal RNA gene expression as RNA loading controls for northern blot analysis of cell lines of varying malignant potential. Analytical Biochemistry, 216, 223–226.
Bond, B. C., Virley, D. J., Cairns, N. J., Hunter, A. J., Moore, G. B. T., Moss, A. W., et al. (2002). The quantification of gene expression in an animal model of brain ischemia using TaqMan real-time RT-PCR. Molecular Brain Research, 106, 101–116.
Bustin, S. A. (2000). Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology, 25, 169–193.
Bustin, S. A. (2002). Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): Trends and problems. Journal of Molecular Endocrinology, 29, 23–39.
Bustin, S. A., & Nolan, T. (2004). Pitfalls of quantitative real-time reverse transcription polymerase chain reaction. Journal of Biomolecular Techniques, 15, 155–166.
Chen, J., Rider, D. A., & Ruan, R. (2006). Identification of valid housekeeping genes and antioxidant enzyme gene expression change in the aging rat liver. Journal of Gerontology, 61A, 20–27.
de Leeuw, W. J., Slagboom, P. E., & Vijg, J. (1989). Quantitative comparison of mRNA levels in mammalian tissues: 28S ribosomal RNA level as an accurate internal control. Nucleic Acids Research, 17, 10137–10138.
Dheda, K., Huggett, J. F., Bustin, S. A., Johnson, M. A., Rook, G., & Zumla, A. (2004). Validation of housekeeping genes for normalizing RNA expression in real-time PCR. BioTechniques, 37, 112–119.
Gatta, L. B., Albertini, A., Ravid, R., & Finazzi, D. (2002). Levels of β-secretase bace and α-secretase adam10 mRNA in Alzheimer hippocampus. Clinical Neuroscience and Neuropathology, 13, 2031–2034.
Gutala, R. V., & Reddy, P. H. (2004). The use of real-time PCR in a gene expression study of Alzheimer’s disease post-mortem brains. Journal of Neuroscience Methods, 132, 101–107.
Krussel, J. S., Huang, H. Y., Simon, C., Behr, B., Pape, A. R., Wen, Y., et al. (1998). Single blastomeres within human preimplantation embryos express different amounts of messenger ribonucleic acid for beta-actin and interleukin-1 receptor type I. Journal of Clinical Endocrinology and Metabolism, 83, 953–959.
Law, A. J., Weickert, C. S., Webster, M. J., Herman, M. M., Kleinman, J. E., & Harrison, P. J. (2003). Changes in NMDA receptor subunit mRNAs and cyclophilin mRNA during development of the human hippocampus. Annals of the New York Academy of Sciences, 1003, 426–430.
Lindblom, J., Haitina, T., Fredriksson, R., & Schioth, H. B. (2005). Differential regulation of nuclear receptors, neuropeptides and peptide hormones in the hypothalamus and pituitary of food restricted rats. Molecular Brain Research, 133, 37–46.
Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using Real-Time quantitative PCR and the 2−ΔΔCT method. Methods, 25, 402–408.
Macdonald, R., Bingham, S., Bond, B. C., Parsons, A. A., & Philpott, K. L. (2001). Determination of changes in mRNA expression in a rat model of neuropathic pain by Taqman quantitative RT-PCR. Molecular Brain Research, 90, 48–56.
Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acid Research, 29, 2002–2007.
Proudnikov, D., Yuferov, V., LaForge, K. S., Ho, A., & Jeanne Kreek, M. (2003). Quantification of multiple mRNA levels in rat brain regions using real time optical PCR. Molecular Brain Research, 112, 182–185.
Roth, C. M. (2002). Quantifying gene expression. Current Issues in Molecular Biology, 4, 93–100.
Sawada, T., Morinobu, S., Tsuji, S., Kawano, K., Watanabe, T., Suenaga, T., et al. (2004). Reduction in levels of amphiphysin 1 mRNA in the hippocampus of aged rats subjected to repeated variable stress. Neuroscience, 126, 461–466.
Schmittgen, T. D., & Zakrajsek, B. A. (2000). Effect of experimental treatment on housekeeping gene expression: Validation by real-time, quantitative RT-PCR. Journal of Biochemical and Biophysical Methods, 46, 69–81.
Shimokawa, I., Fukuyama, T., Yanagihara-Outa, K., Tomita, M., Komatsu, T., Higami, Y., et al. (2003). Effects of caloric restriction on gene expression in the arcuate nucleus. Neurobiology of Aging, 24, 117–123.
Slagboom, P. E., de Leeuw, W. J., & Vijg, J. (1990). Messenger RNA levels and methylation patterns of gapdh and beta-actin genes in rat liver, spleen and brain in relation to aging. Mechanism of Ageing and Development, 53, 243–257.
Sturzenbaum, S. R., & Kille, P. (2001). Control genes in quantitative molecular biological techniques: The variability of invariance. Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology, 130, 281–289.
Tatton, W. G., Chalmers-Redman, R. M., Elstner, M., Leesch, W., Jagodzinski, F. B., Stupak, D. P., et al. (2000). Glyceraldehyde-3-phosphate dehydrogenase in neurodegeneration and apoptosis signaling. Journal of Neural Transmission. Supplementum, 60, 77–100.
Thellin, O., Zorzi, W., Lakaye, B., De Borman, B., Coumans, B., Hennen, G., et al. (1999). Housekeeping genes as internal standards: Use and limits. Journal of Biotechnology, 75, 291–295.
Tichopad, A., Didier, A., & Pfaffl, M. W. (2004). Inhibition of real-time RT-PCR quantification due to tissue-specific contaminants. Molecular and Cellular Probes, 18, 45–50.
Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., et al. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 3, 0034.I-0034.II
Wall, S. J., & Edwards, D. R. (2002). Quantitative reverse transcriptionpolymerase chain reaction (RT-PCR): A comparison of primerdropping, competitive, and real-time RT-PCRs. Analytical Biochemistry, 300, 269–273.
Webster, M. J., Weickert, C. S., Herman, M. M., & Kleinman, J. E. (2002). BDNF mRNA expression during postnatal development, maturation and aging of the human prefrontal cortex. Developmental Brain Research, 139, 139–150.
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Tanic, N., Perovic, M., Mladenovic, A. et al. Effects of aging, dietary restriction and glucocorticoid treatment on housekeeping gene expression in rat cortex and hippocampus—Evaluation by real time RT-PCR. J Mol Neurosci 32, 38–46 (2007). https://doi.org/10.1007/s12031-007-0006-7
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DOI: https://doi.org/10.1007/s12031-007-0006-7