RESUMO
BACKGROUND: Quantitative real-time PCR (qPCR) is one of the most widely used methods to measure gene expression. An important aspect of qPCR data that has been largely ignored is the presence of non-detects: reactions failing to exceed the quantification threshold and therefore lacking a measurement of expression. While most current software replaces these non-detects with a value representing the limit of detection, this introduces substantial bias in the estimation of both absolute and differential expression. Single imputation procedures, while an improvement on previously used methods, underestimate residual variance, which can lead to anti-conservative inference. RESULTS: We propose to treat non-detects as non-random missing data, model the missing data mechanism, and use this model to impute missing values or obtain direct estimates of model parameters. To account for the uncertainty inherent in the imputation, we propose a multiple imputation procedure, which provides a set of plausible values for each non-detect. We assess the proposed methods via simulation studies and demonstrate the applicability of these methods to three experimental data sets. We compare our methods to mean imputation, single imputation, and a penalized EM algorithm incorporating non-random missingness (PEMM). The developed methods are implemented in the R/Bioconductor package nondetects. CONCLUSIONS: The statistical methods introduced here reduce discrepancies in gene expression values derived from qPCR experiments in the presence of non-detects, providing increased confidence in downstream analyses.
