Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue

• Published in: BMC molecular biology Vol. 9; no. 1; p. 46
• Main Authors: Coulson, David TR, Brockbank, Simon, Quinn, Joseph G, Murphy, Suzanne, Ravid, Rivka, Irvine, G Brent, Johnston, Janet A
• Format: Journal Article
• Language: English
• Published: London BioMed Central 06.05.2008; BioMed Central Ltd
• Subjects: Biochemistry; Biomedical and Life Sciences; Brain; Brain - metabolism; Cell Biology; Gene Expression Profiling; Gene Expression Regulation; Genes; Genetic aspects; Humans; Identification and classification; Life Sciences; Nucleic Acid Chemistry; Physiological aspects; Polymerase chain reaction; Reference Standards; Reproducibility of Results; Research Article; Reverse Transcriptase Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction - standards; RNA - metabolism; Software; United Kingdom; Pairwise Variation; Reference Gene; Reference Gene Stability; Dementia With Lewy Body; Candidate Reference Gene
• ISSN: 1471-2199; 1471-2199
• DOI: 10.1186/1471-2199-9-46

Background Studies of gene expression in post mortem human brain can contribute to understanding of the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Quantitative real-time PCR (RT qPCR) is often used to analyse gene expression. The validity of results obtained using RT qPCR is reliant on accurate data normalization. Reference genes are generally used to normalize RT qPCR data. Given that expression of some commonly used reference genes is altered in certain conditions, this study aimed to establish which reference genes were stably expressed in post mortem brain tissue from individuals with AD, PD or DLB. Results The present study investigated the expression stability of 8 candidate reference genes, (ubiquitin C [UBC], tyrosine-3-monooxygenase [YWHAZ], RNA polymerase II polypeptide [RP II], hydroxymethylbilane synthase [HMBS], TATA box binding protein [TBP], β-2-microglobulin [B2M], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], and succinate dehydrogenase complex-subunit A, [SDHA]) in cerebellum and medial temporal gyrus of 6 AD, 6 PD, 6 DLB subjects, along with 5 matched controls using RT qPCR (TaqMan ® Gene Expression Assays). Gene expression stability was analysed using geNorm to rank the candidate genes in order of decreasing stability in each disease group. The optimal number of genes recommended for accurate data normalization in each disease state was determined by pairwise variation analysis. Conclusion This study identified validated sets of mRNAs which would be appropriate for the normalization of RT qPCR data when studying gene expression in brain tissue of AD, PD, DLB and control subjects.