title: Editor's Highlight: Dose-Response Analysis of RNA-Seq Profiles in Archival Formalin-Fixed Paraffin-Embedded Samples.
abstract: Use of archival resources has been limited to date by inconsistent methods for genomic profiling of degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples. RNA-sequencing offers a promising way to address this problem. Here, we evaluated transcriptomic dose responses using RNA-sequencing in paired FFPE and frozen (FROZ) samples from 2 archival studies in mice, one <2 years old and the other >20 years old. Experimental treatments included 3 different doses of di(2-ethylhexyl)phthalate or dichloroacetic acid for the recently archived and older studies, respectively. Total RNA was ribo-depleted and sequenced using the Illumina HiSeq platform. In the recently archived study, FFPE samples had 35% lower total counts compared to FROZ samples but high concordance in fold-change values of differentially expressed genes (DEGs) (r2=0.99), highly enriched pathways (90% overlap with FROZ), and benchmark dose estimates for preselected target genes (<5% difference vs FROZ). In contrast, older FFPE samples had markedly lower total counts (3% of FROZ) and poor concordance in global DEGs and pathways. However, counts from FFPE and FROZ samples still positively correlated (r2= 0.84 across all transcripts) and showed comparable dose responses for more highly expressed target genes. These findings highlight potential applications and issues in using RNA-sequencing data from FFPE samples. Recently archived FFPE samples were highly similar to FROZ samples in sequencing quality metrics, DEG profiles, and dose-response parameters, while further methods development is needed for older lower-quality FFPE samples. This work should help advance the use of archival resources in chemical safety and translational science.
authors: National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina.
authors: Susan D
authors: SD
authors: Hester
authors: NSF International, Ann Arbor, Michigan.
authors: Virunya
authors: V
authors: Bhat
authors: National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina.
authors: Brian N
authors: BN
authors: Chorley
authors: National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina.
authors: Gleta
authors: G
authors: Carswell
authors: Genomics-Bioinformatics, Expression Analysis Genomic Services, Q2 Solutions, Durham, North Carolina.
authors: Wendell
authors: W
authors: Jones
authors: National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina.
authors: Leah C
authors: LC
authors: Wehmas
authors: National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina wood.charles@epa.gov.
authors: Charles E
authors: CE
authors: Wood
mesh_terms: D000818:Animals; D003999:Dichloroacetic Acid / Q000633:toxicity*; D004051:Diethylhexyl Phthalate / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D005404:Fixatives / Q000737:chemistry*; D005557:Formaldehyde / Q000737:chemistry*; D005615:Freezing; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D053263:Gene Regulatory Networks / Q000187:drug effects; D005819:Genetic Markers; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D051379:Mice; D016612:Paraffin Embedding; D020871:RNA Stability; D017423:Sequence Analysis, RNA; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods*; D018675:Toxicity Tests / Q000379:methods*; D059467:Transcriptome / Q000187:drug effects*
chemical_list: D005404:Fixatives; D005819:Genetic Markers; D005557:Formaldehyde; D003999:Dichloroacetic Acid; D004051:Diethylhexyl Phthalate
keywords: FFPE; RNA-sequencing; archival resources.; benchmark dose; dose–response; toxicogenomics