title: Validation of endogenous control reference genes for normalizing gene expression studies in endometrial carcinoma.
abstract: Real-time quantitative RT-PCR (qRT-PCR) is a powerful technique used for the relative quantification of target genes, using reference (housekeeping) genes for normalization to ensure the generation of accurate and robust data. A systematic examination of the suitability of endogenous reference genes for gene expression studies in endometrial cancer tissues is absent. The aims of this study were therefore to identify and evaluate from the thirty-two possible reference genes from a TaqMan(®) array panel their suitability as an internal control gene. The mathematical software packages geNorm qBasePLUS identified Pumilio homolog 1 (Drosophila) (PUM1), ubiquitin C (UBC), phosphoglycerate kinase (PGK1), mitochondrial ribosomal protein L19 (MRPL19) and peptidylpropyl isomerase A (cyclophilin A) (PPIA) as the best reference gene combination, whilst NormFinder identified MRPL19 as the best single reference gene, with importin 8 (IPO8) and PPIA being the best combination of two reference genes. BestKeeper ranked MRPL19 as the most stably expressed gene. In addition, the study was validated by examining the relative expression of a test gene, which encodes the cannabinoid receptor 1 (CB1). A significant difference in CB1 mRNA expression between malignant and normal endometrium using MRPL19, PPIA, and IP08 in combination was observed. The use of MRPL19, IPO8 and PPIA was identified as the best reference gene combination for the normalization of gene expression levels in endometrial carcinoma. This study demonstrates that the arbitrary selection of endogenous control reference genes for normalization in qRT-PCR studies of endometrial carcinoma, without validation, risks the production of inaccurate data and should therefore be discouraged.
authors.affiliation: Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester LE2 7LX, UK.
authors.forename: Thangesweran
authors.initials: T
authors.lastname: Ayakannu
authors.affiliation: Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester LE2 7LX, UK Biosciences, School of Science and Technology, Nottingham Trent University, Clifton Campus, Nottingham, NG11 8NS.
authors.forename: Anthony H
authors.initials: AH
authors.lastname: Taylor
authors.affiliation: Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester LE2 7LX, UK.
authors.forename: Jonathon M
authors.initials: JM
authors.lastname: Willets
authors.affiliation: Histopathology Department, University Hospital of Leicester NHS Trust, Leicester Royal Infirmary, Leicester LE1 5WW, UK.
authors.forename: Laurence
authors.initials: L
authors.lastname: Brown
authors.affiliation: Division of Anaesthetics, Critical Care and Pain Management, Department of Cardiovascular Sciences, University of Leicester, Leicester LE2 7LX, UK.
authors.forename: David G
authors.initials: DG
authors.lastname: Lambert
authors.affiliation: Division of Anaesthetics, Critical Care and Pain Management, Department of Cardiovascular Sciences, University of Leicester, Leicester LE2 7LX, UK.
authors.forename: John
authors.initials: J
authors.lastname: McDonald
authors.affiliation: Gynaecology Oncology Department, University Hospital of Leicester NHS Trust, Leicester Royal Infirmary, Leicester LE1 5WW, UK.
authors.forename: Quentin
authors.initials: Q
authors.lastname: Davies
authors.affiliation: Gynaecology Oncology Department, University Hospital of Leicester NHS Trust, Leicester Royal Infirmary, Leicester LE1 5WW, UK.
authors.forename: Esther L
authors.initials: EL
authors.lastname: Moss
authors.affiliation: Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester LE2 7LX, UK Department of Obstetrics and Gynaecology, Sidra Medical and Research Center, PO Box 26999, Doha, Qatar jck4@le.ac.uk jkonje@sidra.org.
authors.forename: Justin C
authors.initials: JC
authors.lastname: Konje
mesh_terms: D000465:Algorithms; D014408:Biomarkers, Tumor / Q000235:genetics*; D018269:Carcinoma, Endometrioid / Q000235:genetics* / Q000473:pathology; D016022:Case-Control Studies; D021984:Cyclophilin A / Q000235:genetics; D016889:Endometrial Neoplasms / Q000235:genetics* / Q000473:pathology; D005260:Female; D020869:Gene Expression Profiling / Q000592:standards*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D024101:Mitochondrial Proteins / Q000235:genetics; D060787:Neoplasm Grading; D011237:Predictive Value of Tests; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D043884:Receptor, Cannabinoid, CB1 / Q000235:genetics; D012016:Reference Values; D015203:Reproducibility of Results; D012269:Ribosomal Proteins / Q000235:genetics; D012984:Software; D028961:beta Karyopherins / Q000235:genetics
chemical_list: D014408:Biomarkers, Tumor; C571130:CNR1 protein, human; C107282:IPO8 protein, human; C496590:MRPL19 protein, human; D024101:Mitochondrial Proteins; D043884:Receptor, Cannabinoid, CB1; D012269:Ribosomal Proteins; D028961:beta Karyopherins; D021984:Cyclophilin A
keywords: cannabinoid receptor; endogenous control reference genes; endometrial cancer; human; quantitative RT–PCR