High-throughput RNA sequencing (RNA-seq) is now the standard method to determine differential gene expression. Identifying differentially expressed genes crucially depends on estimates of read-count variability. These estimates are typically based on statistical models such as the negative binomial distribution, which is employed by the tools edgeR, DESeq and cuffdiff. Until now, the validity of these models has usually been tested on either low-replicate RNA-seq data or simulations.