title: Using nonlinear least squares to assess relative expression and its uncertainty in real-time qPCR studies.
abstract: Relative expression ratios are commonly estimated in real-time qPCR studies by comparing the quantification cycle for the target gene with that for a reference gene in the treatment samples, normalized to the same quantities determined for a control sample. For the "standard curve" design, where data are obtained for all four of these at several dilutions, nonlinear least squares can be used to assess the amplification efficiencies (AE) and the adjusted ΔΔCq and its uncertainty, with automatic inclusion of the effect of uncertainty in the AEs. An algorithm is illustrated for the KaleidaGraph program.
authors.affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA. Electronic address: joel.tellinghuisen@vanderbilt.edu.
authors.forename: Joel
authors.initials: J
authors.lastname: Tellinghuisen
mesh_terms: D016018:Least-Squares Analysis; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D035501:Uncertainty
keywords: Data analysis; Nonlinear least squares; PCR; Relative expression; Statistical error