Enhanced and controlled chromatin extraction from FFPE tissues and the application to ChIP-seq
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE128202 | GEO | 2019/03/18
REPOSITORIES: GEO
Similar Datasets
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
Project description:Advent of mass spectrometry based proteomics has revolutionized our ability to study proteins from biological specimenin a high-throughput manner. Unlike cell line based studies, biomedical research involving tissue specimen is often challenging due to limited sample availability. In addition, investigation of clinically relevant research questions often requires enormous amount of time for sample collection prospectively. Formalin fixed paraffin embedded (FFPE) archived tissue samples are a rich source of tissue specimen for biomedical research. However, there are several challenges associated with analysing FFPE samples. Protein cross-linking and degradation of proteins particularly affects proteomic analysis We demonstrate that barocycler that uses pressure-cycling technology enables efficient protein extraction and processing of small amounts of FFPE tissue samplesfor proteomic analysis. We identified 3,525 proteins from six 10µm esophageal squamous cell carcinoma (ESCC) tissue sections. Barocycler allows efficient protein extraction and proteolytic digestion of proteins from FFPE tissue sections where conventional methods have limited success
2018-01-08 | PXD006255 | Pride
Project description:MicroRNAs (miRs) are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE) tissue. Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access) platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p<0.00001) and outlines the optimal performance conditions of this platform using clinical FFPE samples. We outline a method of data analysis looking at differences in miR abundance between FFPE and fresh-frozen samples. By dividing the profiled miR into abundance strata of high (Ct<30), medium (30<Ct<35), and low (Ct>35), we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p<0.001), when examining all miRs, regardless of RNA extraction method used. Examining correlation coefficients between FFPE and fresh-frozen samples in terms of miR abundance reveals correlation coefficients of up to 0.32 (low abundance), 0.70 (medium abundance) and up to 0.97 (high abundance). Our study aims to demonstrate the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.
2010-06-24 | GSE22264 | GEO
Project description:Laser-capture microdissection (LCM) allows the visualization and isolation of morphologically distinct subpopulations of cells from heterogeneous tissue specimens. In combination with formalin-fixed and paraffin-embedded (FFPE) tissue it provides a powerful tool for retrospective and clinically relevant studies of tissue proteins in a healthy and diseased context. In this study, we have developed an optimized protocol to facilitate efficient LCM analysis of FFPE tissue specimens. First, we optimized protein extraction from FFPE tissue by comparing different extraction buffers and investigating the influence of immunohistochemical and haematoxylin & eosin staining on proteins. SDS present in the protein extracts was removed with the SP3 digest method, which was modified to improve protein and peptide recoveries. Using a label-free approach protein expression of microdissected samples was compared to intact tissue sections from substantia nigra to evaluate the efficiency of LCM for the purification of small cell populations. The optimized protocol was used to analyse samples containing as few as ~3,000 cells isolated from the substantia nigra, using FFPE tissue. Replicate samples of 15 healthy donors were analysed in five separate TMT10plex batches, resulting in the quantification of >5,600 protein groups.
2020-03-05 | PXD016563 | Pride
Project description:Formalin fixation and paraffin-embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)-based proteomics has so far proven challenging. Here, we describe an MS-based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from pathology glass slides. We demonstrate broad applicability of the workflow to clinical pathology specimens and variable sample amounts, including low-input cancer tissue isolated by laser microdissection. Using state-of-the-art data dependent acquisition (DDA) and data independent (DIA) MS workflows, we consistently quantify a large part of the proteome in 100 min single-run analyses. In an adenoma cohort comprising more than 100 samples, total work up took less than a day. We observed a moderate trend towards lower protein identifications in long-term stored samples (>15 years) but clustering into distinct proteomic subtypes was independent of archival time. Our results underline the great promise of FFPE tissues for patient phenotyping using unbiased proteomics and prove the feasibility of analyzing large tissue cohorts in a robust, timely and streamlined manner.
2020-04-02 | PXD017269 | Pride
Project description:The optimal conditions and procedures for efficient and reproducible protein extraction of FFPE tissues have not yet been standardised and new sensitive techniques are continually being developed and improved upon. To our knowledge, there is no general agreement as to the choice of detergent or buffer system (and/or addition of PEG 20,000) required for efficient and reproducible protein extraction from human FFPE tissues. Moreover, the effect of PEG 20,000 on protein extraction efficiency has not been evaluated using human FFPE colorectal cancer tissues. This study therefore aims to assess the impact of PEG 20,000 on the protein extraction efficiency, reproducibility, and protein selection bias of the protein extraction buffer used for FFPE colonic resection tissue in label-free LC-MS/MS analysis. The sample pellets were also tested for residual protein, not extracted in the initial extraction. The results show that the absence of PEG 20,000 increases the number of peptides and proteins identified by unfractionated LC-MS/MS analysis, and the method is more reproducible. However, no significant differences were observed with regard to protein selection bias. We propose that studies generating high protein yields would benefit from the absence of PEG 20,000 in the protein extraction buffer.
2022-02-15 | PXD014419 | Pride
Project description:For more than a century, Formalin Fixed Paraffin Embedded (FFPE) sample preparation has been the preferred method for long-term preservation of biological material. However, the use of FFPE samples for epigenomic studies has been difficult because of chromatin damage from long exposure to high concentrations of formaldehyde. Previously, we introduced Cleavage Under Targeted Accessible Chromatin (CUTAC), an antibody-targeted chromatin accessibility mapping protocol based on CUT&Tag. Here we show that simple modifications of our single-tube CUTAC protocol are sufficient to produce high-resolution maps of paused RNA Polymerase II (RNAPII) at enhancers and promoters using FFPE samples. We find that transcriptional regulatory element differences produced by FFPE-CUTAC distinguish between mouse brain tumor specimens and identify regulatory element markers with high confidence and precision. Our simple workflow is suitable for automation, making possible affordable epigenomic profiling of archived biological samples for biomarker identification, clinical applications and retrospective studies.
2023-09-08 | GSE235876 | GEO
Project description:Formalin-fixed paraffin embedded (FFPE) is the clinical gold standard of tissue preservation globally. We present a novel method for optimal extraction of proteins from FFPE tissue samples. The method combines non-toxic, proteomic inert mineral oil for deparaffinization, followed by extraction using elevated temperature and pressure (121˚C; 15 psi). Our novel TOP (Temperature, Oil, and Pressure) proteome extraction is four times faster, less laborious and increases the protein yield. Furthermore, the TOP method is environmentally friendly, as opposed to traditional methods. Comparison of extracted protein by mass spectrometry based proteomics revealed that TOP is not sensitive to formalin over-fixation, unlike the traditional approach, a fact that makes it suitable in the clinical setting. We expect that the TOP proteome extraction platform will help to alleviate some of the difficulties proteomics is facing in the clinical setting.
2024-02-12 | PXD002526 | Pride
Project description:Formalin-fixed, paraffin-embedded (FFPE) tissue samples are an invaluable resource to study the underlying molecular mechanisms of the diseases and when coupled with laser capture microdissection (LCM, isolation of sub histological regions of the tissue sections is readily obtained for further analysis. LCM-based FFPE tissue proteomics is gaining clinicopathological significance particularly in biomarker discovery driven research, beyond its conventional morphology-based application in laboratory diagnosis. Processing of laser capture microdissected tissue sections can be challenging for quantitative proteomic analysis due to lower amount of protein retrieved and losses during the sample processing. A robust, streamlined and automated sample preparation workflow for efficient processing of large cohort of LCM samples which is a primary requisite for biomarker type of studies is needed. Here, we propose a new sample processing workflow for processing of FFPE samples and enable scalable, automated extraction of clean peptides from unprocessed or H&E-stained FFPE tissue sections for deep bottom-up protein profiling and quantification.
2023-10-24 | PXD040727 | Pride