title: System-specific periodicity in quantitative real-time polymerase chain reaction data questions threshold-based quantitation.
abstract: Real-time quantitative polymerase chain reaction (qPCR) data are found to display periodic patterns in the fluorescence intensity as a function of sample number for fixed cycle number. This behavior is seen for technical replicate datasets recorded on several different commercial instruments; it occurs in the baseline region and typically increases with increasing cycle number in the growth and plateau regions. Autocorrelation analysis reveals periodicities of 12 for 96-well systems and 24 for a 384-well system, indicating a correlation with block architecture. Passive dye experiments show that the effect may be from optical detector bias. Importantly, the signal periodicity manifests as periodicity in quantification cycle (Cq) values when these are estimated by the widely applied fixed threshold approach, but not when scale-insensitive markers like first- and second-derivative maxima are used. Accordingly, any scale variability in the growth curves will lead to bias in constant-threshold-based Cqs, making it mandatory that workers should either use scale-insensitive Cqs or normalize their growth curves to constant amplitude before applying the constant threshold method.
authors.affiliation: Department of Andrology, University Hospital Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Andrej-Nikolai
authors.initials: AN
authors.lastname: Spiess
authors.affiliation: Institute of Biotechnology, Brandenburg University of Technology, Cottbus - Senftenberg, Senftenberg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Rödiger
authors.affiliation: Department of Genomics, Faculty of Biotechnology, University of Wroclaw, Wrocław, Poland.
authors.forename: Michał
authors.initials: M
authors.lastname: Burdukiewicz
authors.affiliation: Clinic and Polyclinic for Dermatology and Venerology, University Hospital Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Volksdorf
authors.affiliation: Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, USA.
authors.forename: Joel
authors.initials: J
authors.lastname: Tellinghuisen
mesh_terms: D006801:Humans; D008956:Models, Chemical; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*