Research article

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

David TR Coulson¹ , Simon Brockbank¹ , Joseph G Quinn¹ , Suzanne Murphy¹ , Rivka Ravid² , G Brent Irvine¹ and Janet A Johnston¹

¹Division of Psychiatry and Neuroscience, School of Medicine and Dentistry, Queen's University Belfast, Whitla Medical Building, Belfast, BT9 7BL, Northern Ireland, UK

²Netherlands Brain Bank, Amsterdam, The Netherlands

author email corresponding author email

BMC Molecular Biology 2008, 9:46doi:10.1186/1471-2199-9-46

Published:	6 May 2008

Abstract

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.