Case study: Targeted RNA-sequencing of aged formalin-fixed paraffin-embedded samples for understanding chemical mode of action
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 difficu...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-01-01
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| Series: | Toxicology Reports |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2214750022000956 |
| _version_ | 1811188682067017728 |
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| author | Michael D. Cannizzo Charles E. Wood Susan D. Hester Leah C. Wehmas |
| author_facet | Michael D. Cannizzo Charles E. Wood Susan D. Hester Leah C. Wehmas |
| author_sort | Michael D. Cannizzo |
| collection | DOAJ |
| description | 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 (R2 ≥ 0.94). Similarly, high concordance in fold-change values was observed between TempO-Seq FFPE and RNA-Seq FROZ results (R2 ≥ 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. |
| first_indexed | 2024-04-11T14:22:24Z |
| format | Article |
| id | doaj.art-abd933fa85be4600a08e1d6386fa3df4 |
| institution | Directory Open Access Journal |
| issn | 2214-7500 |
| language | English |
| last_indexed | 2024-04-11T14:22:24Z |
| publishDate | 2022-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Toxicology Reports |
| spelling | doaj.art-abd933fa85be4600a08e1d6386fa3df42022-12-22T04:19:00ZengElsevierToxicology Reports2214-75002022-01-019883894Case study: Targeted RNA-sequencing of aged formalin-fixed paraffin-embedded samples for understanding chemical mode of actionMichael D. Cannizzo0Charles E. Wood1Susan D. Hester2Leah C. Wehmas3Center for Computational Toxicology and Exposure, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, NC 27709, USA; Oak Ridge Institute for Science and Education, US Environmental Protection Agency, Research Triangle Park, NC 27709, USACenter for Computational Toxicology and Exposure, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, NC 27709, USACenter for Computational Toxicology and Exposure, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, NC 27709, USACenter for Computational Toxicology and Exposure, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, NC 27709, USA; Correspondence to: U.S. Environmental Protection Agency, Research Triangle Park, MD-B105-03, 109T.W. Alexander Drive, NC, 27709 USA.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 (R2 ≥ 0.94). Similarly, high concordance in fold-change values was observed between TempO-Seq FFPE and RNA-Seq FROZ results (R2 ≥ 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.http://www.sciencedirect.com/science/article/pii/S2214750022000956Formalin-fixed paraffin-embedded (FFPE)RNA-SeqTempO-SeqDichloroacetic acidDCABenchmark dose (BMD) analysis |
| spellingShingle | Michael D. Cannizzo Charles E. Wood Susan D. Hester Leah C. Wehmas Case study: Targeted RNA-sequencing of aged formalin-fixed paraffin-embedded samples for understanding chemical mode of action Toxicology Reports Formalin-fixed paraffin-embedded (FFPE) RNA-Seq TempO-Seq Dichloroacetic acid DCA Benchmark dose (BMD) analysis |
| title | Case study: Targeted RNA-sequencing of aged formalin-fixed paraffin-embedded samples for understanding chemical mode of action |
| title_full | Case study: Targeted RNA-sequencing of aged formalin-fixed paraffin-embedded samples for understanding chemical mode of action |
| title_fullStr | Case study: Targeted RNA-sequencing of aged formalin-fixed paraffin-embedded samples for understanding chemical mode of action |
| title_full_unstemmed | Case study: Targeted RNA-sequencing of aged formalin-fixed paraffin-embedded samples for understanding chemical mode of action |
| title_short | Case study: Targeted RNA-sequencing of aged formalin-fixed paraffin-embedded samples for understanding chemical mode of action |
| title_sort | case study targeted rna sequencing of aged formalin fixed paraffin embedded samples for understanding chemical mode of action |
| topic | Formalin-fixed paraffin-embedded (FFPE) RNA-Seq TempO-Seq Dichloroacetic acid DCA Benchmark dose (BMD) analysis |
| url | http://www.sciencedirect.com/science/article/pii/S2214750022000956 |
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