Journal of Molecular Neuroscience

, Volume 37, Issue 3, pp 238–253 | Cite as

Selection of Reference Genes for Quantitative Real-time RT-PCR Studies in Mouse Brain

  • Enrica Boda
  • Alessandro Pini
  • Eriola Hoxha
  • Roberta Parolisi
  • Filippo Tempia
Article

Abstract

Since a growing number of studies based on the real-time reverse transcriptase polymerase chain reaction (RT-PCR) continue to be published in order to highlight genes specifically involved in brain development, maturation, and function, the identification of reference genes suitable for this kind of experiments is now an urgent need in the neuroscience field. The aim of this work was to verify the suitability of some very common housekeeping genes (such as Gapdh, 18s, and B2m) and of some relatively new control genes (such as Pgk1, Tfrc, and Gusb) during mouse brain maturation. We tested the candidate reference genes in mouse whole brain, cerebellum, brain stem, hippocampus, medial septum, frontal neocortex, and olfactory bulb. Moreover, we reported the first complete study of Pgk1 expression throughout the development and the aging of mouse brain. Although no tested gene showed to be the optimal reference for all mouse brain regions, in general, the new housekeeping genes were highly stable in most of the analyzed regions. Above all, with few exceptions, Pgk1 showed to be a reliable control for the analyzed mouse brain regions during development, maturation, and aging.

Keywords

Housekeeping gene Real-time reverse transcriptase polymerase chain reaction (RT-PCR) Mouse brain Development Aging Stability 

Notes

Acknowledgements

The experiments were supported by grants (to FT) from: MIUR (PRIN-2005), Regione Piemonte (Ricerca Scientifica Applicata 2004 projects A183 and A74 and Ricerca Sanitaria Finalizzata 2006 and 2007), Compagnia di San Paolo, Fondazione CRT (Progetto Alfieri). EB is recipient of a CRT fellowship (Progetto Lagrange). The authors gratefully thank Annarita De Luca for the helpful suggestions and Matteo Novello for the technical support.

Supplementary material

12031_2008_9128_Fig1_ESM.gif (118 kb)
ESM Fig 1

(GIF 121 KB)

12031_2008_9128_Fig1_ESM.tif (3.6 mb)
High-resolution image (TIFF 3.8 MB)
12031_2008_9128_MOESM1_ESM.doc (514 kb)
ESM(DOC 526 KB)

References

  1. 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. doi:10.1158/0008-5472.CAN-04-0496.PubMedCrossRefGoogle Scholar
  2. Bagley, J., Larocca, G., Jimenez, D. A., & Urban, N. N. (2007). Adult neurogenesis and specific replacement of interneuron subtypes in the mouse main olfactory bulb. BMC Neuroscience, 8, 92. doi:10.1186/1471-2202-8-92.PubMedCrossRefGoogle Scholar
  3. Barroso, I., Benito, B., Garcí-Jiménez, C., Hernández, A., Obregón, M. J., & Santisteban, P. (1999). Norepinephrine, tri-iodothyronine and insulin upregulate glyceraldehyde-3-phosphate dehydrogenase mRNA during Brown adipocyte differentiation. European Journal of Endocrinology, 141, 169–179. doi:10.1530/eje.0.1410169.PubMedCrossRefGoogle Scholar
  4. Bond, B. C., Virley, D. J., Cairns, N. J., et al. (2002). The quantification of gene expression in an animal model of brain ischaemia using TaqMan real-time RT-PCR. Brain Research. Molecular Brain Research, 106, 101–116. doi:10.1016/S0169-328X(02)00417-5.PubMedCrossRefGoogle Scholar
  5. Chen, R. W., Saunders, P. A., Wei, H., Li, Z., Seth, P., & Chuang, D. M. (1999). Involvement of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and p53 in neuronal apoptosis: evidence that GAPDH is upregulated by p53. The Journal of Neuroscience, 19, 9654–9662.PubMedGoogle Scholar
  6. Ehninger, D., & Kempermann, G. (2008). Neurogenesis in the adult hippocampus. Cell and Tissue Research, 331, 243–250. doi:10.1007/s00441-007-0478-3.PubMedCrossRefGoogle Scholar
  7. Flanagan, J. M., Rhodes, M., Wilson, M., & Beutler, E. (2006). The identification of a recurrent phosphoglycerate kinase mutation associated with chronic haemolytic anaemia and neurological dysfunction in a family from USA. British Journal of Haematology, 134, 233–237. doi:10.1111/j.1365-2141.2006.06143.x.PubMedCrossRefGoogle Scholar
  8. Goldman-Wohl, D. S., Chan, E., Baird, D., & Heintz, N. (1994). Kv3.3b: a novel Shaw type potassium channel expressed in terminally differentiated cerebellar Purkinje cells and deep cerebellar nuclei. The Journal of Neuroscience, 14, 511–522.PubMedGoogle Scholar
  9. Goldowitz, D., & Hamre, K. (1998). The cells and molecules that make a cerebellum. Trends in Neurosciences, 21, 375–382. doi:10.1016/S0166-2236(98)01313-7.PubMedCrossRefGoogle Scholar
  10. Gutala, R. V., & Reddy, P. H. (2004). The use of real-time PCR analysis in a gene expression study of Alzheimer’s disease post-mortem brains. Journal of Neuroscience Methods, 132, 101–107. doi:10.1016/j.jneumeth.2003.09.005.PubMedCrossRefGoogle Scholar
  11. Ishitani, R., Tanaka, M., Sunaga, K., Katsube, N., & Chuang, D. M. (1998). Nuclear localization of overexpressed glyceraldehyde-3-phosphate dehydrogenase in cultured cerebellar neurons undergoing apoptosis. Molecular Pharmacology, 53, 701–707.PubMedGoogle Scholar
  12. Johansson, S., Fuchs, A., Okvist, A., et al. (2007). Validation of endogenous controls for quantitative gene expression analysis: application on brain cortices of human chronic alcoholics. Brain Research, 1132, 20–28. doi:10.1016/j.brainres.2006.11.026.PubMedCrossRefGoogle Scholar
  13. Jones, D. T., Trowbridge, I. S., & Harris, A. L. (2006). Effects of transferrin receptor blockade on cancer cell proliferation and hypoxia-inducible factor function and their differential regulation by ascorbate. Cancer Research, 66, 2749–2756. doi:10.1158/0008-5472.CAN-05-3857.PubMedCrossRefGoogle Scholar
  14. Jung, M., Ramankulov, A., Roigas, J., et al. (2007). In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR. BMC Molecular Biology, 8, 47. doi:10.1186/1471-2199-8-47.PubMedCrossRefGoogle Scholar
  15. Livak, K. J., & Schmittgen, T. D. (2001). Methods. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Method, 25, 402–408. doi:10.1006/meth.2001.1262.CrossRefGoogle Scholar
  16. Lossi, L., Tamagno, I., & Merighi, A. (2004). Molecular morphology of neuronal apoptosis: analysis of caspase 3 activation during postnatal development of mouse cerebellar cortex. Journal of Molecular Histology, 35, 621–629. doi:10.1007/s10735-004-2189-3.PubMedCrossRefGoogle Scholar
  17. Matsumoto, A., Arai, Y., Urano, A., & Hyodo, S. (1994). Androgen regulates gene expression of cytoskeletal proteins in adult rat motoneurons. Hormones and Behavior, 28, 357–366. doi:10.1006/hbeh.1994.1032.PubMedCrossRefGoogle Scholar
  18. Mescher, A. L., & Munaim, S. I. (1988). Transferrin and the growth-promoting effect of nerves. International Review of Cytology, 110, 1–26. doi:10.1016/S0074-7696(08)61846-X.PubMedCrossRefGoogle Scholar
  19. Paxinos, G., & Franklin, K. B. J. (2001). The mouse brain in stereotaxic coordinates. New York: Academic.Google Scholar
  20. Perrot-Sinal, T. S., Davis, A. M., & McCarthy, M. M. (2001). Developmental sex differences in glutamic acid decarboxylase (GAD(65)) and the housekeeping gene, GAPDH. Brain Research, 922, 201–208. doi:10.1016/S0006-8993(01)03167-5.PubMedCrossRefGoogle Scholar
  21. Pohjanvirta, R., Niittynen, M., Lindén, J., Boutros, P. C., Moffat, I. D., & Okey, A. B. (2006). Evaluation of various housekeeping genes for their applicability for normalization of mRNA expression in dioxin-treated rats. Chemico-Biological Interactions, 160, 134–149. doi:10.1016/j.cbi.2006.01.001.PubMedCrossRefGoogle Scholar
  22. 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. Brain Research. Molecular Brain Research, 112, 182–185. doi:10.1016/S0169-328X(03)00056-1.PubMedCrossRefGoogle Scholar
  23. Rakic, P. (1974). Neurons in rhesus monkey visual cortex: systematic relation between time of origin and eventual disposition. Science, 183, 425–427. doi:10.1126/science.183.4123.425.PubMedCrossRefGoogle Scholar
  24. Rudy, B., & McBain, C. J. (2001). Kv3 channels: voltage-gated K+ channels designed for high-frequency repetitive firing. Trends in Neurosciences, 24, 517–526. doi:10.1016/S0166-2236(00)01892-0.PubMedCrossRefGoogle Scholar
  25. Sacco, T., De Luca, A., & Tempia, F. (2006). Properties and expression of Kv3 channels in cerebellar Purkinje cells. Molecular and Cellular Neurosciences, 33, 170–179. doi:10.1016/j.mcn.2006.07.006.PubMedCrossRefGoogle Scholar
  26. Sawa, A., Khan, A. A., Hester, L. D., & Snyder, S. H. (1997). Glyceraldehyde-3-phosphate dehydrogenase: nuclear translocation participates in neuronal and nonneuronal cell death. Proceedings of the National Academy of Sciences of the United States of America, 94, 11669–11674. doi:10.1073/pnas.94.21.11669.PubMedCrossRefGoogle Scholar
  27. 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. Mechanisms of Ageing and Development, 53, 243–257. doi:10.1016/0047-6374(90)90042-E.PubMedCrossRefGoogle Scholar
  28. Sotelo-Silveira, J., Crispino, M., Puppo, A., Sotelo, J. R., & Koenig, E. (2008). Myelinated axons contain beta-actin mRNA and ZBP-1 in periaxoplasmic ribosomal plaques and depend on cyclic AMP and F-actin integrity for in vitro translation. Journal of Neurochemistry, 104, 545–557.PubMedGoogle Scholar
  29. Svaasand, E. K., Aasly, J., Landsem, V. M., & Klungland, H. (2007). Altered expression of PGK1 in a family with phosphoglycerate kinase deficiency. Muscle & Nerve, 36, 679–684. doi:10.1002/mus.20859.CrossRefGoogle Scholar
  30. Swisshelm, K., Disteche, C. M., Thorvaldsen, J., Nelson, A., & Salk, D. (1990). Age-related increase in methylation of ribosomal genes and inactivation of chromosome-specific rRNA gene clusters in mouse. Mutation Research, 237, 131–146. doi:10.1016/0921-8734(90)90019-N.PubMedCrossRefGoogle Scholar
  31. Tanic, N., Perovic, M., Mladenovic, A., Ruzdijic, S., & Kanazir, S. (2007). Effects of aging, dietary restriction and glucocorticoid treatment on housekeeping gene expression in rat cortex and hippocampus-evaluation by real time RT-PCR. Journal of Molecular Neuroscience, 32, 38–46. doi:10.1007/s12031-007-0006-7.PubMedCrossRefGoogle Scholar
  32. Vandesompele, J., De Preter, K., Pattyn, F., et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, RESEARCH0034.Google Scholar
  33. Wong, C. C., & Leung, M. S. (2001). Effects of neonatal hypothyroidism on the expressions of growth cone proteins and axon guidance molecules related genes in the hippocampus. Molecular and Cellular Endocrinology, 184, 143–150. doi:10.1016/S0303-7207(01)00592-5.PubMedCrossRefGoogle Scholar
  34. Wood, K. A., Dipasquale, B., & Youle, R. J. (1993). In situ labeling of granule cells for apoptosis-associated DNA fragmentation reveals different mechanisms of cell loss in developing cerebellum. Neuron, 11, 621–632. doi:10.1016/0896-6273(93)90074-2.PubMedCrossRefGoogle Scholar
  35. Yang, M. H., Yoo, K. H., Yook, Y. J., et al. (2007). The gene expression profiling in murine cortical cells undergoing programmed cell death (PCD) induced by serum deprivation. Journal of Biochemistry and Molecular Biology, 40, 277–285.PubMedGoogle Scholar
  36. Zhang, H. L., Eom, T., Oleynikov, Y., et al. (2001). Neurotrophin-induced transport of a beta-actin mRNP complex increases beta-actin levels and stimulates growth cone motility. Neuron, 31, 261–275. doi:10.1016/S0896-6273(01)00357-9.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2008

Authors and Affiliations

  • Enrica Boda
    • 1
    • 2
  • Alessandro Pini
    • 1
    • 2
    • 3
  • Eriola Hoxha
    • 1
    • 2
  • Roberta Parolisi
    • 1
    • 2
  • Filippo Tempia
    • 1
    • 2
  1. 1.Section of Physiology of the Department of NeuroscienceUniversity of TorinoTorinoItaly
  2. 2.National Institute of Neuroscience-ItalyTorinoItaly
  3. 3.Department of Anatomy, Histology and Forensic MedicineUniversity of FirenzeFirenzeItaly

Personalised recommendations