Selection of novel reference genes for use in the human central nervous system: a BrainNet Europe Study
- 1.1k Downloads
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.
KeywordsNeurodegeneration 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.
- 2.Albertella MR, Jones H, Thomson W, Olavesen MG, Neville M, Campbell RD (1996) Localisation of eight additional genes in the human major histocompatibility complex, including the gene encoding the casein kinase II beta subunit, and DNA sequence analysis of the class III region. DNA Seq J DNA Seq Mapp 7:9–12Google Scholar
- 8.Bustin SA (2004) A–Z of quantitative PCR. International University Line, La JollaGoogle Scholar
- 11.Cahoy JD, Emery B, Kaushal A, Foo LC, Zamanian JL, Christopherson KS, Xing Y, Lubischer JL, Krieg PA, Krupenko SA, Thompson WJ, Barres BA (2008) A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci 28:264–278PubMedCrossRefGoogle Scholar
- 19.Durrenberger PF, Fernando S, Kashefi SN, Ferrer I, Hauw JJ, Seilhean D, Smith C, Walker R, Al-Sarraj S, Troakes C, Palkovits M, Kasztner M, Huitinga I, Arzberger T, Dexter DT, Kretzschmar H, Reynolds R (2010) Effects of antemortem and postmortem variables on human brain mRNA quality: a BrainNet Europe study. J Neuropathol Exp Neurol 69:70–81PubMedCrossRefGoogle Scholar
- 25.Hsiao LL, Dangond F, Yoshida T, Hong R, Jensen RV, Misra J, Dillon W, Lee KF, Clark KE, Haverty P, Weng Z, Mutter GL, Frosch MP, Macdonald ME, Milford EL, Crum CP, Bueno R, Pratt RE, Mahadevappa M, Warrington JA, Stephanopoulos G, Gullans SR (2001) A compendium of gene expression in normal human tissues. Physiol Genomics 7:97–104PubMedGoogle Scholar
- 33.Lanoix D, Lacasse AA, St-Pierre J, Taylor SC, Ethier-Chiasson M, Lafond J, Vaillancourt C (2012) Quantitative PCR pitfalls: the case of the human placenta. Mol BiotechnolGoogle Scholar
- 49.Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3: RESEARCH0034Google Scholar