RNA-Sequencing (RNA-seq) has emerged as a standard approach for toxicogenomics research. Under the 3-R (reduce, refine, and replace) principles for animal welfare protection, three animals per group are commonly used in current toxicogenomics studies. It is critical to understand how toxicological interpretation of RNA-seq data may be affected by key technical elements of RNA-seq, such as sequencing depth and library construction, when only three biological replicates are used.
Researchers at the National Center for Toxicological Research conducted a comprehensive comparative analysis to address this question using rats treated with aflatoxin b1 (AFB1), a model hepatotoxin and focusing on key mechanisms of AFB1 toxicity. They compared differential gene expression and pathway enrichment in multiple RNA-seq datasets, which were generated from identical samples but with varying sequencing depths and library preparation. A cross-platform analysis was also performed by comparing data from RNA-seq, microarray, TempO-seq, and qPCR using the same samples.