Low RNA yield and quality limit use of formalin-fixed paraffin-embedded (FFPE) tissue samples for genomic analyses. In this study, we evaluated methods to demodify RNA highly fragmented and crosslinked by formalin fixation. Primary endpoints were RNA recovery, RNA-sequencing quality metrics, and target gene responses to a reference chemical (phenobarbital, PB). Frozen mouse liver samples from control and PB groups (n=6/group) were divided and preserved for 3 months as follows: frozen (FR); 70% ethanol (OH); 10% buffered formalin for 18 hours followed by ethanol (18F); and 10% buffered formalin (3F). Samples from OH, 18F, and 3F groups were processed to FFPE blocks and sectioned for RNA isolation. The latter group received no additional treatment (3F) or the following demodification protocols: short heated incubation with TAE buffer; overnight heated incubation with an organocatalyst using two different isolation kits; or overnight heated incubation without organocatalyst. TruSeq Stranded Total RNA libraries with Ribo-Zero were built and sequenced using the Illumina HiSeq platform. Extended incubation with or without organocatalyst increased RNA yield >3-fold and enhanced quality compared to 3F, as indicated by higher RNA integrity number (>1.5-fold) and fragment analysis values (>3.0-fold). Post-sequencing metrics showed reduced bias in gene coverage and deletion rates for all extended incubation groups. Following PB-induced differential gene expression analysis, all demodification groups showed increased overlap with FR in genes (73-83%) and pathways (91-94%) compared to 3F overlap with FR (60% and 63%, respectively). These results demonstrate simple changes in RNA isolation methods that can enhance genomic analyses of FFPE samples. This dataset is associated with the following publication: Wehmas, L., C. Wood, R. Gagne, A. Williams, C. Yauk, M. Gosink, D. Dalmas, R. Hao, R. O'Lone, and S. Hester. Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 162(2): 535-547, (2018).

Low RNA yield and quality limit use of formalin-fixed paraffin-embedded (FFPE) tissue samples for genomic analyses. In this study, we evaluated methods to demodify RNA highly fragmented and crosslinked by formalin fixation. Primary endpoints were RNA recovery, RNA-sequencing quality metrics, and target gene responses to a reference chemical (phenobarbital, PB). Frozen mouse liver samples from control and PB groups (n=6/group) were divided and preserved for 3 months as follows: frozen (FR); 70% ethanol (OH); 10% buffered formalin for 18 hours followed by ethanol (18F); and 10% buffered formalin (3F). Samples from OH, 18F, and 3F groups were processed to FFPE blocks and sectioned for RNA isolation. The latter group received no additional treatment (3F) or the following demodification protocols: short heated incubation with TAE buffer; overnight heated incubation with an organocatalyst using two different isolation kits; or overnight heated incubation without organocatalyst. TruSeq Stranded Total RNA libraries with Ribo-Zero were built and sequenced using the Illumina HiSeq platform. Extended incubation with or without organocatalyst increased RNA yield >3-fold and enhanced quality compared to 3F, as indicated by higher RNA integrity number (>1.5-fold) and fragment analysis values (>3.0-fold). Post-sequencing metrics showed reduced bias in gene coverage and deletion rates for all extended incubation groups. Following PB-induced differential gene expression analysis, all demodification groups showed increased overlap with FR in genes (73-83%) and pathways (91-94%) compared to 3F overlap with FR (60% and 63%, respectively). These results demonstrate simple changes in RNA isolation methods that can enhance genomic analyses of FFPE samples. This dataset is associated with the following publication: Wehmas, L., C. Wood, R. Gagne, A. Williams, C. Yauk, M. Gosink, D. Dalmas, R. Hao, R. O'Lone, and S. Hester. Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 162(2): 535-547, (2018).

RNA sequencing data from paired: (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) tissue samples from the livers of mice exposed to 600 ppm phenobarbital vs. vehicle control for 7 days in drinking water. RNA sequencing data from paired fresh frozen and 18 hr. formalin-fixed paraffin-embedded liver tissue from mice administered 1500, 3000, and 6000 ppm DEHP or vehicle control for 7 days by dietary ingestion; or RNA sequencing data from paired fresh frozen, 18 hr. formalin fixed paraffin embedded, or 3 wk. formalin fixed paraffin embedded liver tissue from mice administered 8 mg/kg/d Furan or vehicle control for 3 weeks by gavage. This dataset is associated with the following publication: Wehmas, L., S. Hester, and C.E. Wood. Direct Formalin Fixation Induces Widespread Transcriptomic Effects in Archival Tissue Samples. Scientific Reports. Nature Publishing Group, London, UK, 10: 14497, (2020).

Gene expression data on >20 year-old, paired frozen and archival FFPE liver samples generated using targeted resequencing (TempO-Seq) and whole-genome RNA-sequencing 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. Portions of this dataset are inaccessible because: Including all of these files on ScienceHub would exceed the 1 Gb limit. They can be accessed through the following means: The data is stored on the L: Drive. Information on all of the files and file paths are indicated in the 20210923_Readme.xlsx file uploaded with the data. Format: Excel files, tab delimited files, csv files, and sequencing FASTQ files. This dataset is associated with the following publication: 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).

Gene expression data on >20 year-old, paired frozen and archival FFPE liver samples generated using targeted resequencing (TempO-Seq) and whole-genome RNA-sequencing 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. Portions of this dataset are inaccessible because: Including all of these files on ScienceHub would exceed the 1 Gb limit. They can be accessed through the following means: The data is stored on the L: Drive. Information on all of the files and file paths are indicated in the 20210923_Readme.xlsx file uploaded with the data. Format: Excel files, tab delimited files, csv files, and sequencing FASTQ files. This dataset is associated with the following publication: 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).

The primary goal of this study was to investigate the effects of whether organocatalyst (ORGΔ) treatment improves DNA-sequencing data by overcoming formalin-induced artifacts from clinical formalin-fixed paraffin-embedded (FFPE) samples. We isolated RNA and DNA ± ORGΔ from paired FFPE and frozen human renal and ovarian carcinoma specimens collected as part of the National Cancer Institute Biospecimen Pre-analytical Variables program. Tumor types were microscopically confirmed from adjacent tissue sections. Following extraction, DNA was fragmented and sequence differences were compared between frozen and FFPE sample pairs. Treatment with ORGΔ improved concurrent SNP calls in FFPE DNA compared to non-ORGΔ FFPE samples and enhanced confidence in SNP calls for all FFPE DNA samples, beyond that of matched frozen samples. In general, the concordant SNPs identified in paired frozen and FFPE DNA samples agreed for both genotype and homozygosity vs. heterozygosity of calls regardless of ORGΔ treatment. The increased confidence in ORGΔ FFPE DNA variant calls relative to the matched frozen DNA suggests a novel application of this method. With further optimization, this method may improve quality of DNA-sequencing data in frozen as well as FFPE tissue samples. These findings could have important implications for studies using FFPE samples, especially if adequate matched controls are not available. This dataset is associated with the following publication: Wehmas, L., C. Wood, P. Guan, M. Gosink, and S. Hester. Organocatalyst treatment improves variant calling and mutant detection in archival clinical samples. Scientific Data. Springer Nature, New York, NY, 12: 6509, (2022).

We evaluated the Templated Oligo with Sequencing Readout (TempO-Seq) assay for High-throughput transcriptomic screening of a small set of environmental chemicals. This assay yields sequencing reads of exactly 50 base pairs that can be rapidly aligned to generate gene counts, and is compatible with cell lysates prepared in multiwell format. The version of the TempO-Seq assay we evaluated provides nearly whole transcriptome coverage (>20,000 genes). This study encompasses 2 replicates of a 384-well plate design. The majority of wells contain U-2 OS cells exposed to 11 test chemicals at 7 different concentrations (two replicate per test chemical, concentration, and plate), and additional reference samples and controls. Controls include DMSO vehicle treatments (22 per plate). Reference samples include bulk lysate MCF7 samples (2 DMSO treated and 2 TSA treated samples per plate), reference chemical treatments (3 chemicals at single conc each, per plate), and vendor-provided reference RNA mixtures (UHRR and HBRR). This dataset is associated with the following publication: Nyffeler, J., C. Willis, D. Harris, L. Taylor, R. Judson, L. Everett, and J. Harrill. Combining phenotypic profiles and targeted RNA-Seq reveals linkages between transcriptional perturbations and chemical effects on cell morphology: retinoic acid as an example.. TOXICOLOGY AND APPLIED PHARMACOLOGY. Academic Press Incorporated, Orlando, FL, USA, 444: 116032, (2022).

This data set is composed of Tables, Figures, zip file containing the whole manuscript individual datasets. This dataset is associated with the following publication: Wehmas, L., C. Wood, B. Chorley, C. Yauk, G. Nelson, and S. Hester. Enhanced Quality Metrics for Assessing RNA Derived From Archival Formalin-Fixed Paraffin-Embedded Tissue Samples. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 170(2): 357-373, (2019).

This data set is composed of Tables, Figures, zip file containing the whole manuscript individual datasets. This dataset is associated with the following publication: Wehmas, L., C. Wood, B. Chorley, C. Yauk, G. Nelson, and S. Hester. Enhanced Quality Metrics for Assessing RNA Derived From Archival Formalin-Fixed Paraffin-Embedded Tissue Samples. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 170(2): 357-373, (2019).

NIST developed a reference material consisting of synthetic fragments of the SARS-CoV-2 virus RNA, which is the target of molecular diagnostic tests. These RNA fragments can assist in the development and validation of RT-qPCR assays for the detection SARS-CoV-2. The RNA fragments are characterized for concentration using digital PCR methods, may be used to assess limits of detection for SARS-CoV-2 assays, and may calibrate other in-house or commercial SARS-CoV-2 controls. This dataset includes data used for RTGM 10169 SARs-Cov-2 Research Grade Test Material validation. The Illumina and ONT sequence data were used to verify the construct sequence.