This study assesses the technical reproducibility and compares the performance of RNA sequencing (RNA-seq) with gene expression arrays. The authors used the Illumina sequencing platform to study mRNA expression levels in liver and kidney RNA samples, aiming to estimate the technical variance and compare its ability to identify differentially expressed genes with existing array technologies. They found that Illumina sequencing data are highly replicable, with minimal technical variation, suggesting that a single lane of sequencing may suffice for many purposes. The information in a single lane of Illumina sequencing data is comparable to that in a single array in enabling the identification of differentially expressed genes, while also allowing for additional analyses such as detection of low-expressed genes, alternative splice variants, and novel transcripts. The study proposes an empirical protocol and a statistical framework for analyzing gene expression using ultra-high-throughput sequencing technology.This study assesses the technical reproducibility and compares the performance of RNA sequencing (RNA-seq) with gene expression arrays. The authors used the Illumina sequencing platform to study mRNA expression levels in liver and kidney RNA samples, aiming to estimate the technical variance and compare its ability to identify differentially expressed genes with existing array technologies. They found that Illumina sequencing data are highly replicable, with minimal technical variation, suggesting that a single lane of sequencing may suffice for many purposes. The information in a single lane of Illumina sequencing data is comparable to that in a single array in enabling the identification of differentially expressed genes, while also allowing for additional analyses such as detection of low-expressed genes, alternative splice variants, and novel transcripts. The study proposes an empirical protocol and a statistical framework for analyzing gene expression using ultra-high-throughput sequencing technology.