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Direct Formalin Fixation Induces Widespread Transcriptomic Effects in Archival Tissue Samples
Citation:
Wehmas, L., S. Hester, AND C. Wood. Direct Formalin Fixation Induces Widespread Transcriptomic Effects in Archival Tissue Samples. Scientific Reports. Nature Publishing Group, London, Uk, 10:14497, (2020). https://doi.org/10.1038/s41598-020-71521-w
Impact/Purpose:
Archival formalin-fixed paraffin-embedded (FFPE) tissue samples hold a wealth of transcriptomic information; however, little is known about potential artifacts. The primary goal of this study was to investigate the effects of formalin fixation on gene expression profiles in archival FFPE samples. Herein we characterized the transcriptional effects of tissue exposed to formalin during the paraffin-embedding process. We show for the first time that formalin effects on total RNA-seq profiles occur at the time fresh tissue is fixed and is abolished by freezing first, suggesting the gene response is indeed biological rather than a post-mortem artifact. Gene profiles resulting from direct fixation indicated disruption of fundamental pathways of cell metabolism, including oxidative stress, mitochondrial dysfunction, and bioenergetic pathways. However, these effects did not confound the detection of chemical treatment-related responses. These findings could have important implications for studies using FFPE samples, especially if adequate matched controls are not available. Our results demonstrate distinct transcriptional effects of formalin fixation that could impact gene expression studies using FFPE samples. More research is needed to better develop methods that may assist in informatically subtracting out the formalin effect when matched FFPE controls are not available.
Description:
Sequencing technologies now provide unprecedented access to genomic information in archival formalin-fixed paraffin-embedded (FFPE) tissue samples. However, little is known about artifacts induced during formalin fixation, which could bias results. Here we evaluated global changes in RNA-sequencing profiles between matched frozen and FFPE samples. RNA-sequencing was performed on liver samples collected from mice treated with 600 ppm of a reference chemical (phenobarbital) or vehicle control for 7 days. Each sample was divided as follows: (1) fresh-frozen, (2) direct-fixed in formalin for 18 hours, (3) frozen then formalin-fixed, or (4) frozen then ethanol-fixed and paraffin-embedded (n=6/group/condition). Direct fixation resulted in 2946 differentially expressed genes (DEGs) vs. fresh-frozen, 98% of which were down-regulated. Freezing prior to formalin fixation had ≥95% fewer DEGs vs. direct fixation, indicating that most formalin-derived transcriptional effects occurred during fixation of fresh tissue. This finding was supported by retrospective studies of paired frozen and FFPE samples, which identified consistent enrichment in oxidative stress, mitochondrial dysfunction, and transcription elongation pathways with direct fixation. Notably, direct formalin fixation in the parent study did not significantly impact response profiles resulting from phenobarbital exposure. Despite this distinct formalin transcriptional effect, our results show that FFPE samples can be used to understand toxicity.
URLs/Downloads:
DOI: Direct Formalin Fixation Induces Widespread Transcriptomic Effects in Archival Tissue Samples
https://pubmed.ncbi.nlm.nih.gov/32879405/