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Selection of novel reference genes for use in the human central nervous system: a BrainNet Europe Study

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Abstract

The use of an appropriate reference gene to ensure accurate normalisation is crucial for the correct quantification of gene expression using qPCR assays and RNA arrays. The main criterion for a gene to qualify as a reference gene is a stable expression across various cell types and experimental settings. Several reference genes are commonly in use but more and more evidence reveals variations in their expression due to the presence of on-going neuropathological disease processes, raising doubts concerning their use. We conducted an analysis of genome-wide changes of gene expression in the human central nervous system (CNS) covering several neurological disorders and regions, including the spinal cord, and were able to identify a number of novel stable reference genes. We tested the stability of expression of eight novel (ATP5E, AARS, GAPVD1, CSNK2B, XPNPEP1, OSBP, NAT5 and DCTN2) and four more commonly used (BECN1, GAPDH, QARS and TUBB) reference genes in a smaller cohort using RT-qPCR. The most stable genes out of the 12 reference genes were tested as normaliser to validate increased levels of a target gene in CNS disease. We found that in human post-mortem tissue the novel reference genes, XPNPEP1 and AARS, were efficient in replicating microarray target gene expression levels and that XPNPEP1 was more efficient as a normaliser than BECN1, which has been shown to change in expression as a consequence of neuronal cell loss. We provide herein one more suitable novel reference gene, XPNPEP1, with no current neuroinflammatory or neurodegenerative associations that can be used for gene quantitative gene expression studies with human CNS post-mortem tissue and also suggest a list of potential other candidates. These data also emphasise the importance of organ/tissue-specific stably expressed genes as reference genes for RNA studies.

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Acknowledgments

We would like to thank all the tissue donors and their families. Also we are grateful to Veronique Sazdovitch and Kasztner Magdolna for technical assistance. We would like also to thank Charles Mein at The Genome Centre (John Vane Science Centre, Queen Mary, University of London, Charterhouse Square, London EC1M 6BQ) for his assistance and expertise. This study was supported by the European Commission under the Sixth Framework Programme (BrainNet Europe II, LSHM-CT-2004-503039). The Multiple Sclerosis and Parkinson’s Disease Tissue Banks at Imperial were supported by the MS Society of Great Britain and Northern Ireland and the Parkinson’s UK respectively.

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Authors and Affiliations

  1. Wolfson Neuroscience Laboratories, Division of Brain Sciences, Imperial College London, Hammersmith Hospital Campus, Burlington Danes Building, Du Cane Road, W12 0NN, London, UK

    Pascal F. Durrenberger, Francisca S. Fernando, Samira N. Kashefi, Federico Roncaroli, David T. Dexter & Richard Reynolds

  2. Department of Cell Biology and Neuroscience, Istituto Superiore di Sanita, Rome, Italy

    Roberta Magliozzi

  3. MAIngenuity Systems Inc, Redwood City, CA, USA

    Timothy P. Bonnert

  4. Institut de Neuropatologia, Idibell, Hospital Universitari de Bellvitge, Universitat de Barcelona, Barcelona, Spain

    Isidro Ferrer

  5. Laboratoire de Neuropathologie, Groupe Hospitalier Pitié-Salpêtrière, CRICM INSERM UMR-S 975, CNRS UMR7225, UPMC, Sorbonne Universités, APHP, Paris, France

    Danielle Seilhean & Brahim Nait-Oumesmar

  6. Centre de Recherche Institut Du Cerveau Et de La Moelle Épinière, Université Pierre et Marie Curie UMR-S975, Inserm U975, Cnrs UMR7725, Paris, France

    Danielle Seilhean & Brahim Nait-Oumesmar

  7. Department of Psychiatry and Psychotherapy, Ludwigs-Maximilians-University Munich, Nußbaumstr. 7, 80336, Munich, Germany

    Andrea Schmitt & Peter Falkai

  8. Medical Faculty Mannheim, Institute of Psychopharmacology, Central Institute of Mental Health, University of Heidelberg, J5, 68159, Mannheim, Germany

    Peter J. Gebicke-Haerter

  9. Department of Psychiatry, Psychosomatic and Psychotherapy, Neurochemistry Laboratory National Parkinson Foundation Centre of Excellence Research Laboratory, University Hospital of Würzburg, Würzburg, Germany

    Edna Grünblatt

  10. Neurobiochemistry Laboratory, Hospital of Child and Adolescent Psychiatry, University of Zürich, Zürich, Switzerland

    Edna Grünblatt

  11. Laboratory of Neuromorphology and Human Brain Tissue Bank, Semmelweis University, Budapest, Hungary

    Miklos Palkovits

  12. IRCCS Istituto Delle Scienze Neurologiche and Dipartimento di Scienze Neurologiche, Università di Bologna, Bologna, Italy

    Piero Parchi & Sabina Capellari

  13. Centre for Neuropathology and Prion Research, Ludwig-Maximilians-University, Munich, Germany

    Thomas Arzberger & Hans Kretzschmar

Authors
  1. Pascal F. Durrenberger
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  2. Francisca S. Fernando
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  3. Roberta Magliozzi
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  4. Samira N. Kashefi
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  5. Timothy P. Bonnert
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  6. Isidro Ferrer
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  7. Danielle Seilhean
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  8. Brahim Nait-Oumesmar
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  9. Andrea Schmitt
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  10. Peter J. Gebicke-Haerter
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  11. Peter Falkai
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  13. Miklos Palkovits
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  14. Piero Parchi
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  15. Sabina Capellari
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  16. Thomas Arzberger
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  17. Hans Kretzschmar
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  18. Federico Roncaroli
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  19. David T. Dexter
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  20. Richard Reynolds
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Correspondence to Richard Reynolds.

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Durrenberger, P.F., Fernando, F.S., Magliozzi, R. et al. Selection of novel reference genes for use in the human central nervous system: a BrainNet Europe Study. Acta Neuropathol 124, 893–903 (2012). https://doi.org/10.1007/s00401-012-1027-z

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