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Enhanced and controlled chromatin extraction from FFPE tissues and the application to ChIP-seq.

ABSTRACT: 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.

SUBMITTER: Zhong J

PROVIDER: S-EPMC6440302 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Enhanced and controlled chromatin extraction from FFPE tissues and the application to ChIP-seq.

Zhong Jian J Ye Zhenqing Z Clark Chad R CR Lenz Samuel W SW Nguyen Justin H JH Yan Huihuang H Robertson Keith D KD Farrugia Gianrico G Zhang Zhiguo Z Ordog Tamas T Lee Jeong-Heon JH

BMC genomics 20190329 1

<h4>Background</h4>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 ef ...[more]

PMID: 30922218

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Project description:BackgroundThe introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation (ChIP) from formalin-fixed paraffin-embedded (FFPE) tissues, has extended the application of chromatin studies to clinical patient samples. However, extensive crosslinking introduced during routine tissue fixation of clinical specimens may hamper the application of PAT-ChIP to genome-wide studies (PAT-ChIP-Seq) from archived tissue samples. The reduced efficiency in chromatin extraction from over-fixed formalin archival samples is the main hurdle to overcome, especially when low abundant epigenetic marks (e.g., H3K4me3) are investigated.ResultsWe evaluated different modifications of the original PAT-ChIP protocol to improve chromatin isolation from FFPE tissues. With this aim, we first made extensive usage of a normal human colon specimen fixed at controlled conditions (24 h, 48 h, and 72 h) to mimic the variability of tissue fixation that is most frequently found in archived samples. Different conditions of chromatin extraction were tested applying either diverse sonication protocols or heat-mediated limited reversal of crosslinking (LRC). We found that, if compared with canonical PAT-ChIP protocol, LRC strongly increases chromatin extraction efficiency, especially when 72-h fixed FFPE samples are used. The new procedure, that we named enhanced PAT-ChIP (EPAT-ChIP), was then applied at genome-wide level using an archival sample of invasive breast carcinoma to investigate H3K4me3, a lowly abundant histone modification, and H3K27me3 and H3K27ac, two additional well-known histone marks.ConclusionsEPAT-ChIP procedure improves the efficiency of chromatin isolation from FFPE samples allowing the study of long time-fixed specimens (72 h), as well as the investigation of low distributed epigenetic marks (e.g., H3K4me3) and the analysis of multiple histone marks from low amounts of starting material. We believe that EPAT-ChIP will facilitate the application of chromatin studies to archived pathology samples, thus contributing to extend the current understanding of cancer epigenomes and enabling the identification of clinically useful tumor biomarkers.

Dataset Information

Enhanced and controlled chromatin extraction from FFPE tissues and the application to ChIP-seq.

Publications

Enhanced and controlled chromatin extraction from FFPE tissues and the application to ChIP-seq.

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