Skip to main content

Advertisement

SpringerLink
Log in

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

  • Published:
Journal of Molecular Neuroscience Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    PubMed  CAS  Google Scholar 

  • Ehninger, D., & Kempermann, G. (2008). Neurogenesis in the adult hippocampus. Cell and Tissue Research, 331, 243–250. doi:10.1007/s00441-007-0478-3.

    Article  PubMed  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • Paxinos, G., & Franklin, K. B. J. (2001). The mouse brain in stereotaxic coordinates. New York: Academic.

    Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    PubMed  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

Download references

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.

Author information

Author notes

  1. Alessandro Pini

    Present address: Department of Anatomy, Histology and Forensic Medicine, University of Firenze, Viale Pieraccini 6, 50139, Firenze, Italy

Authors and Affiliations

  1. Section of Physiology of the Department of Neuroscience, University of Torino, Corso Raffaello 30, 10125, Torino, Italy

    Enrica Boda, Alessandro Pini, Eriola Hoxha, Roberta Parolisi & Filippo Tempia

  2. National Institute of Neuroscience-Italy, Torino, Italy

    Enrica Boda, Alessandro Pini, Eriola Hoxha, Roberta Parolisi & Filippo Tempia

Authors
  1. Enrica Boda
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alessandro Pini
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eriola Hoxha
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Roberta Parolisi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Filippo Tempia
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Enrica Boda.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM Fig 1

(GIF 121 KB)

High-resolution image (TIFF 3.8 MB)

ESM

(DOC 526 KB)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Boda, E., Pini, A., Hoxha, E. et al. Selection of Reference Genes for Quantitative Real-time RT-PCR Studies in Mouse Brain. J Mol Neurosci 37, 238–253 (2009). https://doi.org/10.1007/s12031-008-9128-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12031-008-9128-9

Keywords

Search

Navigation