title: Critical appraisal of quantitative PCR results in colorectal cancer research: can we rely on published qPCR results?
abstract: The use of real-time quantitative polymerase chain reaction (qPCR) in cancer research has become ubiquitous. The relative simplicity of qPCR experiments, which deliver fast and cost-effective results, means that each year an increasing number of papers utilizing this technique are being published. But how reliable are the published results? Since the validity of gene expression data is greatly dependent on appropriate normalisation to compensate for sample-to-sample and run-to-run variation, we have evaluated the adequacy of normalisation procedures in qPCR-based experiments. Consequently, we assessed all colorectal cancer publications that made use of qPCR from 2006 until August 2013 for the number of reference genes used and whether they had been validated. Using even these minimal evaluation criteria, the validity of only three percent (6/179) of the publications can be adequately assessed. We describe common errors, and conclude that the current state of reporting on qPCR in colorectal cancer research is disquieting. Extrapolated to the study of cancer in general, it is clear that the majority of studies using qPCR cannot be reliably assessed and that at best, the results of these studies may or may not be valid and at worst, pervasive incorrect normalisation is resulting in the wholesale publication of incorrect conclusions. This survey demonstrates that the existence of guidelines, such as MIQE, is necessary but not sufficient to address this problem and suggests that the scientific community should examine its responsibility and be aware of the implications of these findings for current and future research.
authors.affiliation: Department of Pathology, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Electronic address: Jeroen.Dijkstra@radboudumc.nl.
authors.forename: J R
authors.initials: JR
authors.lastname: Dijkstra
authors.affiliation: Lady Davis Institute for Medical Research, Jewish General Hospital, LDI-E447, 3755 Cote Ste-Catherine, Montreal, QC, Canada H3T 1E2.
authors.forename: L C
authors.initials: LC
authors.lastname: van Kempen
authors.affiliation: Department of Pathology, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
authors.forename: I D
authors.initials: ID
authors.lastname: Nagtegaal
authors.affiliation: Postgraduate Medical Institute, Faculty of Health, Social Care and Education, Anglia Ruskin University, Bishops Hall Lane, Chelmsford, Essex CM1 1SQ, United Kingdom.
authors.forename: S A
authors.initials: SA
authors.lastname: Bustin
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics; D035843:Biomedical Research / Q000379:methods / Q000592:standards; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D006801:Humans; D011379:Prognosis; D011642:Publications / Q000592:standards; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results
chemical_list: D014408:Biomarkers, Tumor
keywords: Colorectal cancer; MIQE; Real time PCR; qPCR