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Direct Formalin Fixation Induces Widespread Genomic Effects in Archival Tissues
Citation:
Wehmas, L., S. Hester, AND C. Wood. Direct Formalin Fixation Induces Widespread Genomic Effects in Archival Tissues. SOT, san antonia, Texas, March 11 - 13, 2018.
Impact/Purpose:
While archival formalin-fixed paraffin-embedded (FFPE) tissue samples hold huge promise for retrospective mode of action studies, little is known about potential genomic artifacts induced by formalin fixation, which could affect toxicological and clinical studies being conducted in FFPE samples. Here we investigated whether direct contact of live tissue with formalin was initiating a transcriptomic response independent of tissue processing or RNA sequencing. We found that tissue directly fixed with formalin had many differentially expressed genes most of which were down-regulated compared to frozen. Comparative analysis of this formalin-induced gene set with two independent studies in Ingenuity Pathway Analysis identified consistent enrichment in oxidative stress, mitochondrial dysfunction, and transcription elongation pathways lending support to our formalin effect hypothesis. Direct fixation did not have a clear impact on chemical response. Upstream regulator analysis identified receptor activation consistent with our referent chemical indicating the formalin gene signature did not confound the results. These finding highlight distinct transcriptional effects of formalin fixation that could impact RNA-sequencing studies using FFPE samples. Our results will aid in advancing chemical safety through retrospective analysis of FFPE tissue samples for AOP discovery and development.
Description:
Recent advances in next generation sequencing have dramatically improved transcriptional analysis of degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples. However, little is known about potential genomic artifacts induced by formalin fixation, which could affect toxicological and clinical studies being conducted in FFPE samples. Previously, we identified a consistent shift in RNA-sequencing profiles between matching frozen and FFPE samples. We hypothesized that this shift was due to a core set of transcriptional changes induced when fresh tissue is fixed in formalin. To test this idea, liver samples were collected from male B6C3F1 mice treated with 600 ppm of phenobarbital (PB) or vehicle control (Con) for 7 days. Samples were divided into the following conditions:1) fresh-frozen (FR); 2) directly fixed in 10% buffered formalin for 18 hours and processed to FFPE (FIX); and 3) processed as for FIX but initially frozen (FR>FIX) (n=6/group/condition). The FR>FIX group served as a control for tissue processing and sequencing after collection. Total RNA libraries were prepared and ribo-depleted prior to sequencing on an Illumina Hi-seq 2500. Reads were aligned using Star (2.4) and analyzed in Partek Flow (6.0). Direct fixation (FIX vs. FR) resulted in 2946 differentially expressed genes (DEGs), 98% of which were down regulated. Freezing prior to fixation (FR>FIX vs. FR) resulted in 95% fewer DEGs, indicating that the majority of formalin effect occurred at the time of fixation (i.e., as a transcriptional response) rather than during tissue processing or sequencing. Comparative analysis of this formalin-induced gene set with two independent studies in Ingenuity Pathway Analysis identified consistent enrichment in oxidative stress, mitochondrial dysfunction, and transcription elongation pathways. However, direct fixation did not have a clear impact on chemical response. PB treatment induced 180 DEGs within the FIX group and 159 in the FR>FIX group of which 120 were shared. The DEGs in each list were consistent with CAR/PXR activation and PB exposure, suggesting the formalin signature did not confound the chemical response. Our results highlight distinct transcriptional effects of formalin fixation that could impact RNA-sequencing studies using FFPE samples. This abstract does not reflect EPA policy.