RNA Sequencing of Pooled Samples Effectively Identifies Differentially Expressed Genes

Abstract

Analysis of gene expression changes across the genome provides a powerful, unbiased tool for gaining insight into molecular mechanisms. We have effectively used RNA sequencing to identify differentially expressed genes in long-lived genetic mutants in C. elegans to advance our understanding of the genetic pathways that control longevity. Although RNA sequencing costs have come down, cost remains a barrier to examining multiple strains and time points with a sufficient number of biological replicates. To circumvent this, we have examined the efficacy of identifying differentially expressed genes by sequencing a pooled RNA sample from long-lived isp-1 mitochondrial mutant worms. We found that sequencing a pooled RNA sample could effectively identify genes that were found to be significantly upregulated in the two individually sequenced RNA-seq experiments. Finally, we compared the genes significantly upregulated in the two individually sequenced RNA-seq experiments to two previous microarray experiments to come up with a high-confidence list of modulated genes in long-lived isp-1 mutant worms. Overall, this work demonstrates that RNA sequencing of pooled RNA samples can be used to identify differentially expressed genes.