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Effects of formalin-fixation on FAIRE-Seq and MNase-Seq signatures in yeast

ABSTRACT: Formalin induces inter- and intra-molecular crosslinks within exposed cells. This cross-linking can be exploited to characterise chromatin state as in the FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) and MNase (micrococcal nuclease) assays. Our team aims to optimise these assays for application in museum preserved formalin-exposed specimens. To do so, we first sought to understand the effect of prolonged formalin fixation on the read alignment signatures resulting from FAIRE and MNase treatment. Here we cultured yeast (Saccharomyces cerevisiae) under normal and heat-shock conditions then fixed the cells with formalin for 15 minutes, 1 hour, 6 hours, and 24 hours. We found that heavy formalin fixation modulates rather than eliminates signatures of differential chromatin accessibility and enables semi-quantitative estimates of relative gene expression in this yeast model.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE256156 | GEO | 2024/03/01

REPOSITORIES: GEO

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Effects of formalin-fixation on FAIRE-Seq and MNase-Seq signatures in yeast (RNA-Seq)

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Effects of formalin-fixation on FAIRE-Seq and MNase-Seq signatures in yeast

Project description:Effects of formalin-fixation on FAIRE-Seq and MNase-Seq signatures in yeast

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Effects of formalin-fixation on FAIRE-Seq and MNase-Seq signatures in yeast (RNA-Seq)

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Archival chromatin sequencing in formalin-fixed mouse specimens [FAIRE and Mnase]

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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.

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Genome-wide cancer-specific chromatin accessibility patterns derived from archival processed xenograft tumors

Project description:This study introduces the identification of genome-wide cancer-specific chromatin signatures from archival tissues using nanodroplet-assisted FAIRE-seq. Our enhanced cavitation method, a nanodroplet reagent consisting of a lipid shell with a liquid perfluorocarbon core, facilitated the extraction of intact chromatin from FFPE xenograft tissues across 3 cancer types, encompassing 10 cell lines. Chromatin states that influence gene expression and other nuclear processes can be altered in disease. Studying these patterns in tissues has been limited since existing chromatin accessibility assays are ineffective for archival formalin-fixed, paraffin embedded (FFPE) tissues. Inclusion of nanodroplets during the extraction of chromatin from FFPE tissues enhances the recovery of intact accessible and nucleosome-bound chromatin. We demonstrate that the addition of nanodroplets to the chromatin accessibility assay FAIRE (formaldehyde-assisted isolation of regulatory elements), does not affect the accessible chromatin signal. Applying the technique to FFPE human tumor xenografts, we identified tumor-relevant regions of accessible chromatin shared with those identified in primary tumors. Further, we deconvoluted non-tumor signal to identify cellular components of the tumor microenvironment. Incorporation of this method of enhanced cavitation into FAIRE offers the potential for extending chromatin accessibility to clinical diagnosis and personalized medicine, while also enabling the exploration of gene regulatory mechanisms is archival samples.

2021-10-26 | GSE162443 | GEO

Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity

Project description:INTRODUCTION. Fixation with formalin, a widely adopted procedure to preserve tissue samples, leads to extensive degradation of nucleic acids and thereby compromises procedures like microarray-based gene expression profiling. We hypothesized that RNA fragmentation is caused by activation of RNAses during the interval between formalin penetration and tissue fixation. To prevent RNAse activation, a series of tissue samples were kept under-vacuum at 4°C until fixation and then fixed at 4°C, for 24 hours, in formalin followed by 4 hours in ethanol 95%. RESULTS. The cold-fixation (CF) procedure preserved DNA and RNA, so that RNA segments up to 660 bp were efficiently amplified. Histological and immunohistochemical features were fully comparable with those of standard fixation. Microarray-based gene expression profiles were comparable with those obtained on matched frozen samples for probes hybridizing within 700 bases from the reverse transcription start site. In conclusion, CF preserves tissues and nucleic acids, enabling reliable gene expression profiling of fixed tissues. Thirty samples from human cancers processed in different ways before RNA extraction: (i) Fresh-Freezing (FF), (ii) Standard Fixation with formalin (SF), (iii) Cold-Fixation (CF), a new formalin fixation procedure preserving nucleic acids. The first six samples compare CF with SF and FF in one colorectal cancer (CRC) specimen and one breast cancer (BRCa) specimen. The subsequent 24 samples compare only CF with FF in various cancer specimens. Expression data are neither normalized nor backound subtracted to preserve the original signal distribution for each sample, which is essential to compare expression profiles obtained after different tissue preservation methods.

2011-09-04 | E-GEOD-27175 | biostudies-arrayexpress

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