Case study: Targeted RNA-sequencing of aged formalin-fixed paraffin-embedded samples for understanding chemical mode of action. (2022)

• Record Type: Journal Article
• Authors: Cannizzo, Michael D.; Wood, Charles E.; Hester, Susan D.; Wehmas, Leah C.
• Is Part Of: Toxicology reports. Volume 9(2022)
• Page Start: 883
• Page End: 894
• Publication Date: 2022
• Subjects: AIC Akaike Information Criterion -- BMD Benchmark dose -- BMDA traditional benchmark dose based on apical endpoint -- BMDL lower 95% confidence limit of benchmark dose -- BMDU upper 95% confidence limit of benchmark dose -- BMDT transcriptomic benchmark dose -- CPM counts per million -- DCA dichloroacetic acid -- DEG differentially expressed gene -- DOs detector oligos -- FDR false discovery rate -- FFPE formalin-fixed paraffin-embedded -- FROZ frozen -- GO gene ontology -- IRIS Integrated Risk Information System -- NAM new approach method -- Phred score negative log10 of the base-calling error probability -- RIN RNA integrity number -- RNA-Seq RNA-Sequencing -- TempO-Seq Templated Oligo Sequencing; Formalin-fixed paraffin-embedded (FFPE) -- RNA-Seq -- TempO-Seq -- Dichloroacetic acid -- DCA -- Benchmark dose (BMD) analysis; Toxicology -- Periodicals; Clinical toxicology -- Periodicals; Drug-Related Side Effects and Adverse Reactions; Hazardous Substances; Poisoning; Toxicology; Electronic journals; Periodicals; Periodicals; 571.9505
• DOI: 10.1016/j.toxrep.2022.04.012
• Languages: English
• ISSNs: 2214-7500

Abstract

Abstract: Formalin-fixed paraffin-embedded (FFPE) samples are the only remaining biological archive for many toxicological and clinical studies, yet their use in genomics has been limited due to nucleic acid damage from formalin fixation. Older FFPE samples with highly degraded RNA pose a particularly difficult technical challenge. Probe-based targeted sequencing technologies show promise in addressing this issue but have not been directly compared to standard whole-genome RNA-Sequencing (RNA-Seq) methods. In this study, we evaluated dose-dependent transcriptional changes from paired frozen (FROZ) and FFPE liver samples stored for over 20 years using targeted resequencing (TempO-Seq) and whole-genome RNA-Seq methods. Samples were originally collected from male mice exposed to a reference chemical (dichloroacetic acid, DCA) at 0, 198, 313, and 427 mg/kg-day (n = 6/dose) by drinking water for 6 days. TempO-Seq showed high overlap in differentially expressed genes (DEGs) between matched FFPE and FROZ samples and high concordance in fold-change values across the two highest dose levels of DCA vs. control (R 2 ≥ 0.94). Similarly, high concordance in fold-change values was observed between TempO-Seq FFPE and RNA-Seq FROZ results (R 2 ≥ 0.92). In contrast, RNA-Seq FFPE samples showed few overlapping DEGs compared to FROZ RNA-Seq (≤5 for all dose groups). Modeling of DCA-dependent changes in gene sets identified benchmark doses from TempO-Seq FROZ and FFPE samples within 1.4-fold of RNA-Seq FROZ samples (93.9 mg/kg-d), whereas RNA-Seq FFPE samples were 3.3-fold higher (310.3 mg/kg-d). This work demonstrates that targeted sequencing may provide a more robust method for quantifying gene expression profiles from aged archival FFPE samples. Graphical Abstract: ga1 Highlights: Targeted RNA-Seq outperformed traditional RNA-Seq DCA changes in gene expression of >20 yr old FFPE liver samples. Targeted RNA-Seq of FFPE showed improved gene expression concordance and BMD estimates with paired FROZ samples as opposed to RNA-Seq of FFPE. Exon-targeted probes and direct lysis of FFPE samples for targeted RNA-Seq may circumvent some of the fixation-related damage to nucleic acids. Gene set-based BMD estimates from short-term studies may be useful in measuring biologic potency of chemicals in the absence of long-term studies.