Stratified error in the qPCR assays from the statistical point of view

Abstract
Statistical error affects quantitative PCR at various steps of sample processing as well as during preparation of a standard material. Quantification efficiency is most commonly evaluated from the slope of standard curve serial concentrations projected against the cycle of quantification. The efficiency is considered contributing information to accurately calculate gene expression relative to reference sample and/or reference transcript. Yet, what is the reproducibility of the amplification efficiency estimation in an experimental setup commonly used in labs? Experiment design with repeated preparation of the standard curve was used to estimate the uncertainty associated with estimation of amplification efficiency and its impact on the gene regulation factor calculated. We show that the additive error component due to amplification efficiency estimation may deter the overall gene regulation factor significantly. In addition, we show how various sample processing steps affect the uncertainty of the regulation factor.