title: Assessment of brain reference genes for RT-qPCR studies in neurodegenerative diseases.
abstract: Evaluation of gene expression levels by reverse transcription quantitative real-time PCR (RT-qPCR) has for many years been the favourite approach for discovering disease-associated alterations. Normalization of results to stably expressed reference genes (RGs) is pivotal to obtain reliable results. This is especially important in relation to neurodegenerative diseases where disease-related structural changes may affect the most commonly used RGs. We analysed 15 candidate RGs in 98 brain samples from two brain regions from Alzheimer's disease (AD), Parkinson's disease (PD), Multiple System Atrophy, and Progressive Supranuclear Palsy patients. Using RefFinder, a web-based tool for evaluating RG stability, we identified the most stable RGs to be UBE2D2, CYC1, and RPL13 which we recommend for future RT-qPCR studies on human brain tissue from these patients. None of the investigated genes were affected by experimental variables such as RIN, PMI, or age. Findings were further validated by expression analyses of a target gene GSK3B, known to be affected by AD and PD. We obtained high variations in GSK3B levels when contrasting the results using different sets of common RG underlining the importance of a priori validation of RGs for RT-qPCR studies.
authors.affiliation: Research Laboratory for Stereology and Neuroscience, Bispebjerg-Frederiksberg Hospital, University Hospital of Copenhagen, Bispebjerg Bakke 23, DK-2400, Copenhagen, Denmark.
authors.forename: Rasmus
authors.initials: R
authors.lastname: Rydbirk
authors.affiliation: Research Laboratory for Stereology and Neuroscience, Bispebjerg-Frederiksberg Hospital, University Hospital of Copenhagen, Bispebjerg Bakke 23, DK-2400, Copenhagen, Denmark.
authors.forename: Jonas
authors.initials: J
authors.lastname: Folke
authors.affiliation: Bispebjerg Movement Disorder Biobank, Bispebjerg-Frederiksberg Hospital, University Hospital of Copenhagen, Bispebjerg Bakke 23, DK-2400, Copenhagen, Denmark.|Department of Neurology, Bispebjerg-Frederiksberg Hospital, University Hospital of Copenhagen, Bispebjerg Bakke 23, DK-2400, Copenhagen, Denmark.|Institute of Clinical Medicine, Faculty of Health, University of Copenhagen, Blegdamsvej 3B, DK-2200, Copenhagen, Denmark.
authors.forename: Kristian
authors.initials: K
authors.lastname: Winge
authors.affiliation: Research Laboratory for Stereology and Neuroscience, Bispebjerg-Frederiksberg Hospital, University Hospital of Copenhagen, Bispebjerg Bakke 23, DK-2400, Copenhagen, Denmark.
authors.forename: Susana
authors.initials: S
authors.lastname: Aznar
authors.affiliation: Research Laboratory for Stereology and Neuroscience, Bispebjerg-Frederiksberg Hospital, University Hospital of Copenhagen, Bispebjerg Bakke 23, DK-2400, Copenhagen, Denmark.|Institute of Clinical Medicine, Faculty of Health, University of Copenhagen, Blegdamsvej 3B, DK-2200, Copenhagen, Denmark.
authors.forename: Bente
authors.initials: B
authors.lastname: Pakkenberg
authors.affiliation: Research Laboratory for Stereology and Neuroscience, Bispebjerg-Frederiksberg Hospital, University Hospital of Copenhagen, Bispebjerg Bakke 23, DK-2400, Copenhagen, Denmark.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Brudek
mesh_terms: D001921:Brain / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics; D019636:Neurodegenerative Diseases / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D009419:Nerve Tissue Proteins