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| Status |
Public on Sep 08, 2020 |
| Title |
Direct Formalin Fixation Induces Widespread Transcriptomic Effects in Archival Tissue Samples |
| Organisms |
Mus musculus; Rattus norvegicus |
| Experiment type |
Expression profiling by high throughput sequencing
|
| Summary |
Archival formalin-fixed paraffin-embedded (FFPE) tissue samples hold a wealth of transcriptomic information; however, little is known about potential artifacts. Previously, we identified a consistent shift in global RNA-sequencing profiles between matching frozen and FFPE samples. We hypothesized that this shift was from fixing fresh tissue in formalin. To test this idea, RNA-sequencing was performed on liver samples collected from male mice treated with 600 ppm of a reference chemical (phenobarbital, 600 ppm phenobarbital) or vehicle control for 7 days. Samples were divided into: (1) fresh-frozen (FR); (2) directly fixed in 10% buffered formalin for 18 hours followed by paraffin embedding (FFPE); (3) frozen then fixed as FFPE (FR>FFPE); or (4) frozen then fixed in 70% ethanol followed by paraffin embedding (FR>OH) (n=6/group/condition). Direct fixation resulted in 2946 differentially expressed genes (DEGs), 98% of which were down-regulated. Freezing prior to fixation resulted in ≥95% fewer DEGs vs. FR, indicating that most formalin-derived transcriptional effects occurred with fixation. This was supported by follow-up studies, which identified consistent enrichment in oxidative stress, mitochondrial dysfunction, and transcription elongation pathways with formalin fixation. Notably, formalin fixation in the parent study did not significantly impact chemical response profiles, which were consistent with CAR/PXR activation and 600 ppm phenobarbital exposure. Our results demonstrate distinct transcriptional effects of formalin fixation that could impact gene expression studies using FFPE samples.
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| Overall design |
RNA-sequencing was performed on liver samples collected from male mice treated with 600 ppm of a reference chemical (phenobarbital, 600 ppm phenobarbital) or vehicle control for 7 days. Samples were divided into: (1) fresh-frozen (FR); (2) directly fixed in 10% buffered formalin for 18 hours followed by paraffin embedding (FFPE); (3) frozen then fixed as FFPE (FR>FFPE); or (4) frozen then fixed in 70% ethanol followed by paraffin embedding (FR>OH) (n=6/group/condition).
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| Contributor(s) |
Wehmas L, Hester SD, Wood CE |
| Citation(s) |
32879405 |
| Submission date |
Apr 06, 2020 |
| Last update date |
Sep 08, 2020 |
| Contact name |
Susan Hester |
| E-mail(s) |
hester.susan@epa.gov
|
| Phone |
919-541-1320
|
| Organization name |
US EPA
|
| Street address |
109 TW Alexander Dr
|
| City |
RTP |
| State/province |
NC |
| ZIP/Postal code |
27711 |
| Country |
USA |
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| Platforms (3) |
| GPL17021 |
Illumina HiSeq 2500 (Mus musculus) |
| GPL21626 |
NextSeq 550 (Mus musculus) |
| GPL25029 |
NextSeq 550 (Rattus norvegicus) |
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| Samples (96)
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| Relations |
| BioProject |
PRJNA643500 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE148174_study1_mouse_liver_cpm_normalized_log2transformed_count.txt.gz |
2.3 Mb |
(ftp)(http) |
TXT |
| GSE148174_study1_mouse_liver_raw_count.txt.gz |
1.6 Mb |
(ftp)(http) |
TXT |
| GSE148174_study3_rat_liver_cpm_normalized_log2transformed_count.csv.gz |
237.0 Kb |
(ftp)(http) |
CSV |
| GSE148174_study3_rat_liver_raw_count.csv.gz |
666.0 Kb |
(ftp)(http) |
CSV |
| GSE148174_study3_rat_lung_cpm_normalized_log2transformed_count.csv.gz |
265.6 Kb |
(ftp)(http) |
CSV |
| GSE148174_study3_rat_lung_raw_count.csv.gz |
671.5 Kb |
(ftp)(http) |
CSV |
| GSE148174_study4_AML12_mouse_liver_cpm_normalized_log2transformed_count.txt.gz |
82.6 Kb |
(ftp)(http) |
TXT |
| GSE148174_study4_AML12_mouse_liver_raw_count.txt.gz |
478.2 Kb |
(ftp)(http) |
TXT |
| Processed data are available on Series record |
| Raw data not provided for this record |
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