title: Comparison of microarrays and RNA-seq for gene expression analyses of dose-response experiments.
abstract: Relative to microarrays, RNA-seq has been reported to offer higher precision estimates of transcript abundance, a greater dynamic range, and detection of novel transcripts. However, previous comparisons of the 2 technologies have not covered dose-response experiments that are relevant to toxicology. Male F344 rats were exposed for 13 weeks to 5 doses of bromobenzene, and liver gene expression was measured using both microarrays and RNA-seq. Multiple normalization methods were evaluated for each technology, and gene expression changes were statistically analyzed using both analysis of variance and benchmark dose (BMD). Fold-change values were highly correlated between the 2 technologies, whereas the -log p values showed lower correlation. RNA-seq detected fewer statistically significant genes at lower doses, but more significant genes based on fold change except when a negative binomial transformation was applied. Overlap in genes significant by both p value and fold change was approximately 30%-40%. Random sampling of the RNA-seq data showed an equivalent number of differentially expressed genes compared with microarrays at ~5 million reads. Quantitative RT-PCR of differentially expressed genes uniquely identified by each technology showed a high degree of confirmation when both fold change and p value were considered. The mean dose-response expression of each gene was highly correlated between technologies, whereas estimates of sample variability and gene-based BMD values showed lower correlation. Differences in BMD estimates and statistical significance may be due, in part, to differences in the dynamic range of each technology and the degree to which normalization corrects genes at either end of the scale.
authors.affiliation: * The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709;
authors.forename: Michael B
authors.initials: MB
authors.lastname: Black
authors.forename: Bethany B
authors.initials: BB
authors.lastname: Parks
authors.forename: Linda
authors.initials: L
authors.lastname: Pluta
authors.forename: Tzu-Ming
authors.initials: TM
authors.lastname: Chu
authors.forename: Bruce C
authors.initials: BC
authors.lastname: Allen
authors.forename: Russell D
authors.initials: RD
authors.lastname: Wolfinger
authors.forename: Russell S
authors.initials: RS
authors.lastname: Thomas
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001969:Bromobenzenes / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D020869:Gene Expression Profiling / Q000379:methods*; D008099:Liver / Q000187:drug effects*; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D051381:Rats; D011916:Rats, Inbred F344; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018570:Risk Assessment; D017423:Sequence Analysis, RNA / Q000379:methods*; D043922:Toxicogenetics; D059467:Transcriptome / Q000187:drug effects*
chemical_list: D001969:Bromobenzenes; C032036:bromobenzene
keywords: RNA-seq; RT-PCR; bioinformatics; dose response; microarray; risk assessment.; toxicogenomics