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Case study: Targeted RNA-sequencing of aged formalin-fixed paraffin-embedded samples for understanding chemical mode of action
Citation:
Cannizzo, M., C. Wood, S. Hester, AND L. Wehmas. Case study: Targeted RNA-sequencing of aged formalin-fixed paraffin-embedded samples for understanding chemical mode of action. Toxicology Reports. Elsevier B.V., Amsterdam, Netherlands, 9:883-894, (2022). https://doi.org/10.1016/j.toxrep.2022.04.012
Impact/Purpose:
Archived tissue samples from toxicology studies are a valuable resource for understanding how chemicals cause adverse biological effects across several levels of biological organization (from genes to animals) without the need for new experiments. Nucleic acid damage resulting from preserving tissue in formalin has limited the use of archived tissue in genomics. However, advances in technology, like targeted RNA-sequencing, are making it possible to obtain better gene expression information from these old samples. However, to gain acceptance, this new technology needs to be tested. The present study compared targeted RNA-seq (TempO-Seq) of >20-year-old frozen and matched archival formalin-fixed paraffin-embedded (FFPE) liver tissue samples to previously collected gene expression data using a gold standard method: RNA-sequencing, which worked well with frozen tissue but performed poorly with the paired FFPE tissue samples. Samples were collected from male mice exposed to a well-studied chemical, dichloroacetic acid, at 0, 198, 313, and 427 mg/kg-day, (n=6/dose) by drinking water for 6 days. As proof of concept, changes in gene response from DCA exposure (dose) was compared to existing cellular and 2 year animal effects using benchmark dose (BMD) response modeling to determine how well gene response informed chemical risk vs. traditional animal data, which often takes longer to measure. TempO-Seq of frozen and paired FFPE liver tissue samples identified significant genes and similar changes in gene expression to that of frozen RNA-sequencing results. BMDs from measuring gene-set response in TempO-Seq frozen and FFPE tissue samples were within 1.4-fold of that for RNA-sequencing of frozen samples (93.9 mg/kg-d) and all three methods were within 3.1-fold of the 2 year benchmark dose based on liver adenoma and carcinoma results used in the IRIS risk assessment of DCA. The benchmark dose based on RNA-sequencing of FFPE tissue samples was less sensitive at 310.3 mg/kg-d. This work shows short term gene response can set chemical risk thresholds similar to long term animal studies and that targeted sequencing may provide a more robust method for understanding chemical effects on gene response in decades old archival FFPE tissue samples.
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.
URLs/Downloads:
DOI: Case study: Targeted RNA-sequencing of aged formalin-fixed paraffin-embedded samples for understanding chemical mode of action
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