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Targeted RNA-sequencing of aged archival formalin-fixed paraffin-embedded tissue samples

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 variability in RNA quality from formalin fixation. Older FFPE samples, which often have highly degraded RNA, pose a particularly difficult technical challenge. Recent developments in probe-based targeted sequencing technologies show promise in addressing this issue, while also being more cost effective than traditional RNA-sequencing (RNA-seq) methods. In the current study, we evaluated dose-dependent transcriptional changes from paired frozen and archival 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 flash-frozen (FROZ) samples and concordance in fold-changes across the highest two dose levels of DCA vs. control (R2 ≥ 0.94). Similarly, high concordance in fold-changes was observed between TempO-Seq FFPE and RNA-seq FROZ results (R2 ≥ 0.92). Only 28 DEGs or fewer were identified in RNA-seq FFPE samples. Modeling of DCA-dependent changes in gene sets identified benchmark doses from TempO-Seq FROZ and FFPE samples that were 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE186113 | GEO | 2022/10/10

REPOSITORIES: GEO

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Project description:Nafion byproduct 2 (NBP2; CAS: 749836-20-2; Product #: 6164-3-3J; Lot: 512400; SynQuest Laboratories Alachua, FL, USA) is a polyfluoroalkyl ether sulfonic acid that was recently detected in surface water, drinking water, and human serum samples from monitoring studies in North Carolina, USA. We orally exposed pregnant Sprague-Dawley rats to NBP2 from gestation day (GD) 14–18 (0.1–30 mg/kg/d), GD17-21, and GD8 to postnatal day (PND) 2 (0.3–30 mg/kg/d) to characterize maternal, fetal, and postnatal effects. GD14-18 exposures were also conducted with perfluorooctane sulfonate (PFOS) for comparison to NBP2, as well as data previously published for hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX). NBP2 produced stillbirth (30 mg/kg), reduced pup survival shortly after birth (10 mg/kg), and reduced pup body weight (10 mg/kg). Histopathological evaluation identified reduced glycogen stores in newborn pup livers and hepatocyte hypertrophy in maternal livers at ≥ 10 mg/kg. Exposure to NBP2 from GD14-18 reduced maternal serum total T3 and cholesterol concentrations (30 mg/kg). Maternal, fetal, and neonatal liver gene expression was investigated using RT-qPCR pathway arrays, while maternal and fetal livers were also analyzed using TempO-Seq transcriptomic profiling. Overall, there was limited alteration of genes in maternal or F1 livers from NBP2 exposure with significant changes mostly occurring in the top dose group (30 mg/kg) associated with lipid and carbohydrate metabolism. Metabolomic profiling indicated elevated maternal bile acids for NBP2, but not HFPO-DA or PFOS, while all three reduced 3-indolepropionic acid. Maternal and fetal serum and liver NBP2 concentrations were similar to PFOS, but ∼10–30-fold greater than HFPO-DA concentrations at a given maternal oral dose. NBP2 is a developmental toxicant in the rat, producing neonatal mortality, reduced pup body weight, reduced pup liver glycogen, reduced maternal thyroid hormones, and altered maternal and offspring lipid and carbohydrate metabolism similar to other studied PFAS, with oral toxicity for pup loss that is slightly less potent than PFOS but more potent than HFPO-DA.

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Targeted RNA-sequencing of aged archival formalin-fixed paraffin-embedded tissue samples

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