The Prostate Gene Expression Analysis in Prostate Cancer: The Importance of the Endogenous Control Alice Vajda, 1 * Laure Marignol, 1,2 Ciara Barrett, 3 Stephen F. Madden, 4 Thomas H. Lynch, 5 Donal Hollywood, 1 and Antoinette S. Perry 1 1 Prostate Molecular Oncology, Academic Unit of Clinical and Molecular Oncology, Institute of Molecular Medicine, Trinity College Dublin, Ireland 2 Division of RadiationTherapy, Institute of Molecular Medicine,Trinity College Dublin, Ireland 3 Department of Histopathology, St. James’s Hospital, Dublin, Ireland 4 MolecularTherapeutics for Cancer Ireland, National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland 5 Department of Urology, St. James’s Hospital, Dublin, Ireland BACKGROUND. Aberrant gene expression is a hallmark of cancer. Quantitative reverse- transcription PCR (qRT-PCR) is the gold-standard for quantifying gene expression, and com- monly employs a house-keeping gene (HKG) as an endogenous control to normalize results; the choice of which is critical for accurate data interpretation. Many factors, including sample type, pathological state, and oxygen levels influence gene expression including putative HKGs. The aim of this study was to determine the suitability of commonly used HKGs for qRT-PCR in prostate cancer. METHODS. Prostate cancer (LNCaP, 22Rv1, PC3, and DU145) and normal (PWR1E and RWPE1) cell lines were cultured in air and hypoxia. The performance of 16 HKGs was assessed using Normfinder and coefficient of variation. In silico promoter analysis was per- formed to identify putative hypoxia response elements (HREs). The impact of the endoge- nous control on expression levels of HIF1A and GSTP1 was investigated by qRT-PCR in cell lines and tissue specimens respectively. RESULTS. Hypoxia altered expression of several HKGs: IPO8, B2M, and PGK1. The most stably expressed HKGs were ACTB, PPIA, and UBC. Both UBC and ACTB showed constitu- tive expression of HIF1A in air and hypoxia, while PGK1 falsely implied a sixfold hypoxia- induced down-regulation. In prostate tumors, UBC and PGK1 both revealed down-regulation of GSTP1 relative to matched benign, whereas ACTB showed variability. CONCLUSIONS. This study demonstrates that no universal endogenous control exists for gene expression studies, even within one disease type. It highlights the importance of validating expression of intended HKGs between different sample types and environmental exposures. Prostate # 2012 Wiley Periodicals, Inc. KEY WORDS: qRT-PCR; house-keeping gene; prostate cancer; endogenous control INTRODUCTION Quantitative or real-time reverse transcription PCR (qRT-PCR) is a commonly used laboratory technique in cancer research, enabling the sensitive quantifica- tion of gene expression levels [1]. However, it holds a number of potential pitfalls, one of which is normali- zation of gene expression results. This is of para- mount importance, as there are multiple factors related to the processes of RNA extraction, reverse transcription, and PCR that can introduce variation Additional supporting information may be found in the online ver- sion of this article. Grant sponsor: Irish Health Research Board; Grant sponsor: Irish Cancer Society; Grant sponsor: Prostate Cancer Foundation. *Correspondence to: Alice Vajda, Prostate Molecular Oncology, Institute of Molecular Medicine, St. James’s Hospital, Room 2.20, Dublin 8, Ireland. E-mail: vajdaa@tcd.ie Received 7 June 2012; Accepted 2 August 2012 DOI 10.1002/pros.22578 Published online in Wiley Online Library (wileyonlinelibrary.com). ß 2012 Wiley Periodicals, Inc.