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Direct formalin fixation induces widespread transcriptomic effects in archival tissue samples.

ABSTRACT: 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 a reference chemical (phenobarbital) or vehicle control for 7 days. Each sample was divided into four parts: (1) fresh-frozen, (2) direct-fixed in formalin for 18 h, (3) frozen then formalin-fixed, and (4) frozen then ethanol-fixed and paraffin-embedded (n = 6/group/condition). Direct fixation resulted in 2,946 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 in the liver occurred during fixation. This finding was supported by retrospective studies of paired frozen and FFPE samples, which identified consistent enrichment in oxidative stress, mitochondrial dysfunction, and transcription initiation pathways with direct fixation. Notably, direct formalin fixation in the parent study did not significantly impact response profiles resulting from chemical exposure. These results advance our understanding of FFPE samples as a resource for genomic research.

SUBMITTER: Wehmas LC

PROVIDER: S-EPMC7468282 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Direct Formalin Fixation Induces Widespread Transcriptomic Effects in Archival Tissue Samples

Project description: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.

2020-09-08 | GSE148174 | GEO

Direct Formalin Fixation Induces Widespread Transcriptomic Effects in Archival Tissue Samples

Project description:Direct Formalin Fixation Induces Widespread Transcriptomic Effects in Archival Tissue Samples

| PRJNA643500 | ENA

Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples.

Project description:Archival formalin-fixed paraffin-embedded (FFPE) tissue samples offer a vast but largely untapped resource for genomic research. The primary technical issues limiting use of FFPE samples are RNA yield and quality. 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 transcriptional 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 h followed by ethanol (18F); or 10% buffered formalin (3F). Samples from OH, 18F, and 3F groups were processed to FFPE blocks and sectioned for RNA isolation. Additional sections from 3F received the following demodification protocols to mitigate RNA damage: short heated incubation with Tris-Acetate-EDTA buffer; overnight heated incubation with an organocatalyst using 2 different isolation kits; or overnight heated incubation without organocatalyst. Ribo-depleted, stranded, total RNA libraries were built and sequenced using the Illumina HiSeq 2500 platform. Overnight incubation (± organocatalyst) increased RNA yield >3-fold and RNA integrity numbers and fragment analysis values by > 1.5- and >3.0-fold, respectively, versus 3F. Postsequencing metrics also showed reduced bias in gene coverage and deletion rates for overnight incubation groups. All demodification groups had increased overlap for differentially expressed genes (77%-84%) and enriched pathways (91%-97%) with FR, with the highest overlap in the organocatalyst groups. These results demonstrate simple changes in RNA isolation methods that can enhance genomic analyses of FFPE samples.

| S-EPMC6800129 | biostudies-literature

Detection and Genotyping of Human Papillomaviruses from Archival Formalin-Fixed Tissue Samples.

Project description:Pathology departments routinely process and store formalin-fixed, paraffin-embedded (FFPE) tissue samples for clinical diagnosis. These collections often contain decades' worth of samples and represent a treasure trove of specimens that can be analyzed for retrospective epidemiological studies, diagnostics, and pathogen discovery. Accurate amplification and sequencing of DNA from these samples is critical for the usability of these FFPE samples. Here we present a collection of protocols that describe extraction of DNA from FFPE tissues, PCR amplification of human papillomavirus DNA, and subsequent genotyping of the infecting virus. © 2016 by John Wiley & Sons, Inc.

| S-EPMC5117989 | biostudies-literature

Enhanced Quality Metrics for Assessing RNA Derived From Archival Formalin-Fixed Paraffin-Embedded Tissue Samples.

Project description:Formalin-fixed paraffin-embedded (FFPE) tissues provide an important resource for toxicogenomic research. However, variability in the integrity or quality of RNA obtained from archival FFPE specimens can lead to unreliable data and wasted resources, and standard protocols for measuring RNA integrity do not adequately assess the suitability of FFPE RNA. The main goal of this study was to identify improved methods for evaluating FFPE RNA quality for whole-genome sequencing. We examined RNA quality metrics conducted prior to RNA-sequencing in paired frozen and FFPE samples with varying levels of quality based on age in block and time in formalin. RNA quality was measured by the RNA integrity number (RIN), a modified RIN called the paraffin-embedded RNA metric, the percentage of RNA fragments >100-300 nucleotides in size (DV100-300), and 2 quantitative PCR-based methods. This information was correlated to sequencing read quality, mapping, and gene detection. Among fragmentation-based methods, DV and PCR-based metrics were more informative than RIN or paraffin-embedded RNA metric in determining sequencing success. Across low- and high-quality FFPE samples, a minimum of 80% of RNA fragments >100 nucleotides (DV100 > 80) provided the best indication of gene diversity and read counts upon sequencing. The PCR-based methods further showed quantitative reductions in amplifiable RNA of target genes related to sample age and time in formalin that inform input quantity of FFPE RNA for sequencing. These results should aid in screening and prioritizing archival FFPE samples for retrospective analyses of gene expression.

| S-EPMC7029400 | biostudies-literature

Formalin tissue fixation biases myelin-sensitive MRI.

Project description:PurposeChemical fixatives such as formalin form cross-links between proteins and affect the relaxation times and diffusion properties of tissue. These fixation-induced changes likely also affect myelin density measurements produced by quantitative magnetization transfer and myelin water imaging. In this work, we evaluate these myelin-sensitive MRI methods for fixation-induced biases.MethodsWe perform quantitative magnetization transfer, myelin water imaging, and deuterium oxide-exchanged zero TE imaging on unfixed human spinal cord tissue at 9.4 Tesla and repeat these measurements after 1 day and 31 days of formalin fixation.ResultsThe quantitative magnetization-transfer bound pool fraction increased by 30.7% ± 21.1% after 1 day of fixation and by 42.6% ± 33.9% after 31 days of fixation. Myelin water fraction increased by 39.7% ± 15.5% and 37.0% ± 15.9% at these same time points, and mean T2 of the myelin water pool nearly doubled. Reference-normalized deuterium oxide-exchanged zero TE signal intensity increased by 8.17% ± 6.03% after 31 days of fixation but did not change significantly after 1 day of fixation. After fixation, specimen cross-sectional area decreased by approximately 5%; after correction for shrinkage, changes in deuterium oxide-exchanged zero TE intensity were nearly eliminated.ConclusionBound pool fraction and myelin water fraction are significantly increased by formalin fixation, whereas deuterium oxide-exchanged zero TE intensity is minimally affected. Changes in quantitative magnetization transfer and myelin water imaging may be due in part to delamination and formation of vacuoles in the myelin sheath. Deuterium oxide-exchanged signal intensity may be altered by fixation-induced changes in myelin lipid solid-state 1 H T1 . We urge caution in the comparison of these measurements across subjects or specimens in different states, especially unfixed versus fixed tissue.

| S-EPMC6626568 | biostudies-literature

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

Project description: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.

2022-10-10 | GSE186113 | GEO

Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples

Project description: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.

2018-02-28 | GSE103440 | GEO

Multi-platform microRNA profiling of hepatoblastoma patients using formalin fixed paraffin embedded archival samples.

Project description:BackgroundFormalin fixed paraffin embedded (FFPE) samples are a valuable resource in cancer research and have the potential to be extensively used. However, they are often underused because of degradation and chemical modifications occurring in the RNA that can present obstacles in downstream analysis. In routine medical care, FFPE material is examined and archived, therefore clinical collections of many types of cancers exist. It is beneficial to assess and record the quality of data that can be obtained from this type of material. The current study investigated three independent platforms and their ability to profile microRNAs (miRNAs) within FFPE samples from hepatoblastoma (HB) patients.FindingsHere we present three types of datasets consisting of miRNA profiles for 13 HB patients with different tumour types and molecular variations. The three platforms that were used to generate these data are: next-generation sequencing (Illumina MiSeq), microarray (Affymetrix(®) GeneChip(®) miRNA 3.0 array) and NanoString (nCounter, Human v2 miRNA Assay). The mature miRNAs identified are based on miRBase version 17 and 18.ConclusionsThese datasets provide a global landscape of miRNA expression for a rare childhood cancer that has not previously been well characterised. These data could serve as a resource for future studies aiming to make comparisons of HB miRNA profiles and to document aberrant miRNA expression in this type of cancer.

| S-EPMC4660849 | biostudies-literature

Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue.

Project description:Formalin-fixed, paraffin-embedded tissue is the most widely available material for retrospective clinical studies. In combination with the potential of genomics, these tissues represent an invaluable resource for the elucidation of disease mechanisms and validation of differentially expressed genes as novel therapeutic targets or prognostic indicators. We describe here an approach that, in combination with laser-assisted microdissection allows quantitative gene expression analysis in formalin-fixed, paraffin-embedded archival tissue. Using an optimized RNA microscale extraction procedure in conjunction with real-time quantitative reverse transcriptase-polymerase chain reaction based on fluorogenic TaqMan methodology, we analyzed the expression of a panel of cancer-relevant genes, EGF-R, HER-2/neu, FGF-R4, p21/WAF1/Cip1, MDM2, and HPRT and PGK as controls. We demonstrate that expression level determinations from formalin-fixed, paraffin-embedded tissues are accurate and reproducible. Measurements were comparable to those obtained with matching fresh-frozen tissue and neither fixation grade nor time significantly affected the results. Laser microdissection studies with 5-microm thick sections and defined numbers of tumor cells demonstrated that reproducible quantitation of specific mRNAs can be achieved with only 50 cells. We applied our approach to HER-2/neu quantitative gene expression analysis in 54 microdissected tumor and nonneoplastic archival samples from patients with Barrett's esophageal adenocarcinoma and showed that the results matched those obtained in parallel by fluorescence in situ hybridization and immunohistochemistry. Thus, the combination of laser-assisted microdissection and real-time TaqMan reverse transcriptase-polymerase chain reaction opens new avenues for the investigation and clinical validation of gene expression changes in archival tissue specimens.

| S-EPMC1850313 | biostudies-literature

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