Acta Neuropathologica

, Volume 124, Issue 6, pp 893–903 | Cite as

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

  • Pascal F. Durrenberger
  • Francisca S. Fernando
  • Roberta Magliozzi
  • Samira N. Kashefi
  • Timothy P. Bonnert
  • Isidro Ferrer
  • Danielle Seilhean
  • Brahim Nait-Oumesmar
  • Andrea Schmitt
  • Peter J. Gebicke-Haerter
  • Peter Falkai
  • Edna Grünblatt
  • Miklos Palkovits
  • Piero Parchi
  • Sabina Capellari
  • Thomas Arzberger
  • Hans Kretzschmar
  • Federico Roncaroli
  • David T. Dexter
  • Richard ReynoldsEmail author
Methods Paper


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.


Neurodegeneration Validation Normalisation Gene expression studies Post-mortem tissue Internal controls 



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.

Supplementary material

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Supplementary material 1 (XLSX 18 kb)
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Supplementary material 2 (XLSX 6,364 kb)
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Supplementary material 3 (PDF 294 kb)
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Supplementary material 4 (PDF 73 kb)


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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Pascal F. Durrenberger
    • 1
  • Francisca S. Fernando
    • 1
  • Roberta Magliozzi
    • 2
  • Samira N. Kashefi
    • 1
  • Timothy P. Bonnert
    • 3
  • Isidro Ferrer
    • 4
  • Danielle Seilhean
    • 5
    • 6
  • Brahim Nait-Oumesmar
    • 5
    • 6
  • Andrea Schmitt
    • 7
  • Peter J. Gebicke-Haerter
    • 8
  • Peter Falkai
    • 7
  • Edna Grünblatt
    • 9
    • 10
  • Miklos Palkovits
    • 11
  • Piero Parchi
    • 12
  • Sabina Capellari
    • 12
  • Thomas Arzberger
    • 13
  • Hans Kretzschmar
    • 13
  • Federico Roncaroli
    • 1
  • David T. Dexter
    • 1
  • Richard Reynolds
    • 1
    Email author
  1. 1.Wolfson Neuroscience Laboratories, Division of Brain SciencesImperial College LondonLondonUK
  2. 2.Department of Cell Biology and NeuroscienceIstituto Superiore di SanitaRomeItaly
  3. 3.MAIngenuity Systems IncRedwood CityUSA
  4. 4.Institut de NeuropatologiaIdibell, Hospital Universitari de Bellvitge, Universitat de BarcelonaBarcelonaSpain
  5. 5.Laboratoire de Neuropathologie, Groupe Hospitalier Pitié-SalpêtrièreCRICM INSERM UMR-S 975, CNRS UMR7225, UPMC, Sorbonne Universités, APHPParisFrance
  6. 6.Centre de Recherche Institut Du Cerveau Et de La Moelle ÉpinièreUniversité Pierre et Marie Curie UMR-S975, Inserm U975, Cnrs UMR7725ParisFrance
  7. 7.Department of Psychiatry and PsychotherapyLudwigs-Maximilians-University MunichMunichGermany
  8. 8.Medical Faculty Mannheim, Institute of PsychopharmacologyCentral Institute of Mental Health, University of HeidelbergMannheimGermany
  9. 9.Department of Psychiatry, Psychosomatic and PsychotherapyNeurochemistry Laboratory National Parkinson Foundation Centre of Excellence Research Laboratory, University Hospital of WürzburgWürzburgGermany
  10. 10.Neurobiochemistry LaboratoryHospital of Child and Adolescent Psychiatry, University of ZürichZürichSwitzerland
  11. 11.Laboratory of Neuromorphology and Human Brain Tissue BankSemmelweis UniversityBudapestHungary
  12. 12.IRCCS Istituto Delle Scienze Neurologiche and Dipartimento di Scienze NeurologicheUniversità di BolognaBolognaItaly
  13. 13.Centre for Neuropathology and Prion ResearchLudwig-Maximilians-UniversityMunichGermany

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