Enhanced and controlled chromatin extraction from FFPE tissues and the application to ChIP-seq
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ABSTRACT:
PROVIDER: PRJNA526727 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8717010_1.fastq.gz | Fastqsanger.gz | |||
| SRR8717010_2.fastq.gz | Fastqsanger.gz | |||
| SRR8717020_1.fastq.gz | Fastqsanger.gz | |||
| SRR8717020_2.fastq.gz | Fastqsanger.gz | |||
| SRR8717030_1.fastq.gz | Fastqsanger.gz |
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Project description:Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology samples, and thus FFPE samples are a major resource of samples in clinical research. However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples, and are hampered by low chromatin yield in FFPE samples due to the lack of a reliable and efficient chromatin preparation method. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays.This study provided a new method that achieves efficient extraction of high-quality chromatin suitable for chromatin-based epigenetic assays with less damage on chromatin.
2019-03-18 | GSE128202 | GEO
Project description:BACKGROUND:Epigenetic dysregulation is involved in the etiology and progression of various human diseases. Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology samples, and thus FFPE samples are a major resource of samples in clinical research. However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples, and are hampered by low chromatin yield in FFPE samples due to the lack of a reliable and efficient chromatin preparation method. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays. RESULTS:During rehydration of FFPE tissues, applying a tissue-level cross-link reversal into the deparaffinized tissue at 65 °C dramatically increased chromatin yield in the soluble fraction. The resulting chromatin is compatible with targeted ChIP-qPCR and genome-wide ChIP-seq approaches. The chromatin prepared by Chrom-EX PE showed a gradual fragmentation pattern with varying incubation temperature. At temperatures below 37 °C, the majority of soluble chromatin is over 1 kb. The soluble chromatin prepared in the range of 45-60 °C showed a typical nucleosomal pattern. And the majority of chromatin prepared at 65 °C is close to mononucleosomal size. These observations indicate that chromatin preparation from FFPE samples can be controlled for downstream chromatin-based epigenetic assays. CONCLUSIONS:This study provided a new method that achieves efficient extraction of high-quality chromatin suitable for chromatin-based epigenetic assays with less damage on chromatin. This approach may provide a way to circumvent the over-fixed nature of FFPE tissues for future technology development.
| S-EPMC6440302 | biostudies-literature
Project description:The vast majority of tissue biopsies from patients in hospital environments are included in paraffin (FFPE- formalin-fixed and paraffin-embedded) for long-term storage. This fixation process produces a modification in the proteins called "crosslinks", which improves protein stability, necessary for their conservation. Currently, these samples are mainly used in clinical practice for performing immunohistochemical techniques, since these modifications do not suppose a drawback for this technique; however, crosslinks makes the protein extraction process a challenging one. Accordingly, these modifications make the development of a good protein extraction protocol necessary. Due to the specific characteristics of each tissue, the same extraction buffers or deparaffinization protocols are not equally effective in all cases. Therefore, it is necessary to obtain a specific protocol for each tissue. This work aims to establish a deparaffinization and protein extraction protocol from paraffin-embedded kidney samples, which finally allows us to obtain high quantity and quality protein, suitable for the application of proteomic techniques. To achieve this; we have designed different protocols using a range of buffers with diverse compositions and characteristics. Our results showed that xylene can eliminate the formalin fixation from the kidney tissue. Moreover, strong detergents (SDS-sodium dodecil sulfate- or analogues) and reducing agents (e.g. DTT- Dithiothreitol-) in the extraction buffer appear to be more important to obtain a good protein extraction yields and a good number of proteins identifications by LC-MSMS.
2021-09-10 | PXD023891 | Pride
Project description:Objectives: Formalin-fixed paraffin-embedded (FFPE) tissue is the standard material for di-agnostic pathology but poses relevant hurdles to accurate protein extraction due to cross-linking and chemical alterations. While numerous extraction pro-tocols and chemicals have been described, systematic comparative analyses are limited. Various parameters were thus investigated in their qualitative and quantitative effects on protein extraction (PE) efficacy. Special emphasis was put on preservation of membrane proteins (MP) as key subgroup of func-tionally relevant proteins. Methods: Using the example of urothelial carcinoma, FFPE tissue sections were subjected to various deparaffinization, protein extraction and antigen retrieval protocols and buffers as well as different extraction techniques. Performance was meas-ured by protein concentration and western blot analysis of cellular compart-ment markers as well as liquid chromatography-coupled mass spectrometry (LC-MS). Results: Commercially available extraction buffers showed reduced extraction of MPs and came at considerably increased costs. On-slide extraction did not improve PE whereas several other preanalytical steps could be simplified. Systematic variation of temperature and exposure duration demonstrated a quantitatively relevant corridor of optimal antigen retrieval. Conclusions: Preanalytical protein extraction can be optimized at various levels to improve unbiased protein extraction and to reduce time and costs.
2022-05-19 | PXD029133 | Pride
Project description:The study aimed to apply 95GC, originally developed using fresh‑frozen (FF) tissues, to formalin‑fixed paraffin‑embedded (FFPE) tissues, because FFPE tissues are routinely prepared and are readily available. Although we previously reported the applicability of 72GC to FFPE tissues (DOI: 10.1016/j.clbc.2013.11.006), the present study aimed to improve the accuracy of 95GC for FFPE tissues using the reference robust multiarray average (refRMA) method, optimized for FFPE tissues. Therefore, a 95GC RS (Recurrence Score) was first developed and then the accuracy of the newly developed 95GC algorithm for FFPE tissues was evaluated using the 95GC RS. These 14 pairs of FF and FFPE data are included in the concordance analysis of 95GC high/low results between FF and FFPE (Figure 3B). J05 was created in a form more suited to the actual clinical setting, such as leaving the postoperative samples for 4 hours before immersing them in formalin. A long time passed, and now it is unclear how these 14 cases were distributed among the analysis. *Note: This old data has been updated multiple times by the other members. Then, there are some differences from the original paper and unclear points still remain. Therefore, do not use it for formal analysis aimed at public insurance coverage etc. This is for research purposes only. Please cite this paper when writing a new paper. PMID: 31638234 DOI: 10.3892/or.2019.7358
2023-07-11 | GSE236723 | GEO
Project description:Chromatin immunoprecipitation (ChIP) has allowed the study of protein-DNA interactions in chromatin, bringing new insights into the role of histone post-translational modifications (HPTMs) in cancer and other diseases. The coupling of ChIP with next-generation sequencing (NGS) techniques has revealed the distribution of HPTMs over the entire genome allowing the identification of altered epigenetic profiles in pathological conditions. However, until recently, these studies have been limited to cultured cells or fresh/frozen tissues. The development of a new technique named PAT-ChIP has enabled chromatin studies in formalin-fixed paraffin-embedded (FFPE) tissues, opening the doors to a huge number of samples stored in pathology archives and related clinical information. However, chromatin studies in FFPE archival samples can be hindered by the extensive formalin fixation to which these tissues are routinely subjected. Here, we describe enhanced PAT-ChIP (EPAT-ChIP), a new ChIP procedure that facilitates epigenomic studies in archival FFPE tissues. EPAT-ChIP improves the extraction of soluble chromatin, allowing the study of multiple histone marks from limited amounts of starting material. In addition, EPAT-ChIP assures higher yields of final immunoselected DNA, especially when poorly represented histone marks (e.g. narrow peaks of H3K4me3) are investigated, and the consequent production of reliable NGS libraries. In this procedure, FFPE samples are subjected to heat-mediated limited reversal of crosslinking (LRC) which predisposes the tissue to better release soluble chromatin after sonication without altering the antigenic potential of its biomolecules. After investigating differentially-fixed normal colon specimens specifically produced for this study, ePAT-ChIP was successfully used to study three different HPTMs (H3K4me3, K3K27me3, H3K27ac) at genome-wide level in an archival invasive breast carcinoma (IBC) sample.
2018-11-20 | GSE117752 | GEO
Project description:This study was performed to evaluated RNA extraction and gene expression analysis of FFPE specimen stored for more than 20 years. Using long time stored FFPE material; large retrospective studies correlating molecular features with therapeutic response and clinical outcome, can be performed. Quantitative PCR (qPCR) was used to evaluate RNA extraction methods and to compare gene expression profiles of FFPE and fresh frozen (FF) tissue. Extracted RNA was subsequently subjected to microarray analysis and compared to qPCR data. The Ambion RecoverAll kit appears to be particularly suited for RNA extraction of long time stored FFPE tissues. Gene expression analysis using Affymetrix platform displayed a high degree of correlation for endogenous control genes comparing FF and FFPE tissues. We conclude that high quality gene expression signatures can be recognized using Affymetrix gene expression platform on FFPE tissue stored for more than 20 years. However, a general interpretation must be done with caution as different FFPE procedures have varying effects on RNA quality.
2011-09-15 | GSE31735 | GEO
Project description:protein extraction from FFPE tissue comparison
2011-05-05 | PRD000234 | 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
Project description:Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for retrospective studies but protein extraction and subsequent sample processing steps have shown to be challenging for mass spectrometry (MS) analysis. Streamlined high-throughput sample preparation workflows are essential for efficient peptide extraction from complex clinical specimens such as fresh frozen tissues or FFPE. Overall, proteome analysis has gained significant improvements in the instrumentation, acquisition methods, sample preparation workflows and analysis pipelines yet even the most recent FFPE workflows remain complex and are not readily scalable. Here, we present an optimized workflow for Automated Sonication-free Acid-assisted Proteome (ASAP) extraction from FFPE sections. ASAP enables efficient protein extraction from FFPE specimens achieving similar proteome coverage as established methods using time in equipment-heavy sonication-based methods at reduced sample processing time. The broad applicability of ASAP on archived pediatric tumor FFPE specimens resulted in high-quality data with increased proteome coverage and quantitative reproducibility. Our study demonstrates the practicality and superiority of the ASAP workflow as a streamlined, time and cost-effective pipeline for high-throughput FFPE proteomics of clinical specimens.
2024-05-21 | PXD036910 | Pride