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Dose-Response Analysis of RNA-Seq Profiles in Archival Formalin-Fixed Paraffin-Embedded (FFPE) Samples.
Hester, S., V. Bhat, B. Chorley, G. Carswell, W. Jones, L. Wehmas, AND C. Wood. Dose-Response Analysis of RNA-Seq Profiles in Archival Formalin-Fixed Paraffin-Embedded (FFPE) Samples. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 154(2):202-213, (2016).
Biorepositories have immense potential value in characterizing molecular targets of environmental chemicals. However, use of archival resources has been limited to date by inconsistent methods for genomic profiling from formalin-fixed paraffin-embedded (FFPE) samples. RNA-sequencing (RNA-seq) offers an important new way to address this problem. The goal of this study was to evaluate dose-response metrics for RNA-seq data generated from FFPE samples. Our results highlight potential applications and issues in using RNA-sequencing data from FFPE samples. Broadly, this information should be useful to scientists in industry and regulatory settings involved in pathway development and evaluation. These findings should also inform new ways to access different biorepository samples, which could provide a rich bioinformatics pipeline for environmental science and chemical safety.
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 two 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 (2% 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.
Record Details:Record Type: DOCUMENT (JOURNAL/PEER REVIEWED JOURNAL)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL HEALTH AND ENVIRONMENTAL EFFECTS RESEARCH LABORATORY
INTEGRATED SYSTEMS TOXICOLOGY DIVISION