title: Reference genes for reverse transcription quantitative PCR in canine brain tissue.
abstract: BACKGROUND
  In the last decade canine models have been used extensively to study genetic causes of neurological disorders such as epilepsy and Alzheimer's disease and unravel their pathophysiological pathways. Reverse transcription quantitative polymerase chain reaction is a sensitive and inexpensive method to study expression levels of genes involved in disease processes. Accurate normalisation with stably expressed so-called reference genes is crucial for reliable expression analysis.
  
  
  RESULTS
  Following the minimum information for publication of quantitative real-time PCR experiments precise guidelines, the expression of ten frequently used reference genes, namely YWHAZ, HMBS, B2M, SDHA, GAPDH, HPRT, RPL13A, RPS5, RPS19 and GUSB was evaluated in seven brain regions (frontal lobe, parietal lobe, occipital lobe, temporal lobe, thalamus, hippocampus and cerebellum) and whole brain of healthy dogs. The stability of expression varied between different brain areas. Using the GeNorm and Normfinder software HMBS, GAPDH and HPRT were the most reliable reference genes for whole brain. Furthermore based on GeNorm calculations it was concluded that as little as two to three reference genes are sufficient to obtain reliable normalisation, irrespective the brain area.
  
  
  CONCLUSIONS
  Our results amend/extend the limited previously published data on canine brain reference genes. Despite the excellent expression stability of HMBS, GAPDH and HRPT, the evaluation of expression stability of reference genes must be a standard and integral part of experimental design and subsequent data analysis.
authors.affiliation: Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, PO Box 80154, 3508 TD, Utrecht, The Netherlands. Q.E.M.Stassen@uu.nl.
authors.forename: Quirine E M
authors.initials: QE
authors.lastname: Stassen
authors.affiliation: Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, PO Box 80154, 3508 TD, Utrecht, The Netherlands. f.m.riemers@uu.nl.
authors.forename: Frank M
authors.initials: FM
authors.lastname: Riemers
authors.affiliation: Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, PO Box 80154, 3508 TD, Utrecht, The Netherlands. h.a.reijmerink@gmail.com.
authors.forename: Hannah
authors.initials: H
authors.lastname: Reijmerink
authors.affiliation: Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, PO Box 80154, 3508 TD, Utrecht, The Netherlands. p.a.j.leegwater@uu.nl.
authors.forename: Peter A J
authors.initials: PA
authors.lastname: Leegwater
authors.affiliation: Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, PO Box 80154, 3508 TD, Utrecht, The Netherlands. l.c.penning@uu.nl.
authors.forename: Louis C
authors.initials: LC
authors.lastname: Penning
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D002531:Cerebellum / Q000378:metabolism; D004285:Dogs / Q000235:genetics*; D005260:Female; D005625:Frontal Lobe / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D006624:Hippocampus / Q000378:metabolism; D008297:Male; D009778:Occipital Lobe / Q000378:metabolism; D009928:Organ Specificity / Q000235:genetics*; D010296:Parietal Lobe / Q000378:metabolism; D012015:Reference Standards; D013702:Temporal Lobe / Q000378:metabolism; D013788:Thalamus / Q000378:metabolism; D059467:Transcriptome / Q000235:genetics*