Single cell chromatin accessiblity profiling in formlin fixed paraffin embedded samples
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE299388 | GEO | 2025/10/14
REPOSITORIES: GEO
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Project description:For the effectiveness of targeted immunotherapy, which is currently one of the most promising treatments for brain tumors, it is necessary to know specific tumor-associated antigens (TAA). This project aimed to validate the suitability of formalin-fixed and paraffin-embedded (FFPE) material instead of fresh-frozen material for RNA sequencing and downstream TAA identification. Purpose: Detect potential oligodendroglioma-associated antigens using both fresh-frozen and FFPE brain tissues; assess the suitability of the FFPE material for TAA identification. Results: A comparative analysis based on the RNA-seq of canine oligodendroglioma showed that formalin fixation of the samples had a significant effect on the RNA-seq library in terms of quality. Read distribution analysis showed that the FFPE samples contained fewer reads mapping to exonic regions and were enriched with reads mapped to introns and intergenic regions, while the fresh-frozen samples were mostly enriched in reads mapping to exons. Mismatch profile and SNVs calling analyses revealed some substitution artefacts present in the FFPE samples and absent in the corresponding fresh-frozen samples. However, according to the Principal Component Analysis, 36% of the variance between the samples could be explained by the types of conservation, while 43% of the variance could still be attributed to different expression profiles between oligodendroglioma and control conditions. A differential expression analysis of fresh-frozen oligodendroglioma versus fresh-frozen control samples identified 62 potential TAA strongly up-regulated in tumor tissue. The same analysis using the FFPE samples showed 80% of these genes (49/62) also to be differentially expressed. Comparative analysis showed good agreement between FFPE and fresh-frozen RNA-seq libraries in terms of the accuracy of gene expression measurements indicating that archived FFPE tissue can be used for identification of TAA candidates by RNA-seq providing a wealthy source of clinical samples for research. The following 10 genes encoding cell surface proteins have been identified by both approaches as potential TAA candidates based on their strong overexpression in oligodendrogliomas: PDGFRA, NOTCH1, DLL1, GPER1, TNR, IQGAP3, CD44, ERBB3, BCAS1 and ROR2. Future projects still need to investigate their suitability as TAA for targeted immunotherapy. Conclusion: Formalin fixation of the samples had a significant impact on the RNA-seq quality in terms of the accuracy of gene expression measurements, as well as the quality of the nucleotide sequences. Nevertheless, the comparative analysis showed good agreement between FFPE and fresh-frozen RNA-seq libraries.
2020-10-27 | GSE160124 | GEO
Project description:Current spatial transcriptomic methods have been widely used to resolve gene expression; however, these methods are limited to fresh or fresh-frozen samples due to unsuccess of oligo(dT) primers in degraded RNA samples. Here we develop a spatial random-sequencing (spRandom-seq) technology for Formalin-fixed paraffin-embedded (FFPE) tissues by capturing full-length total RNAs with random primers. This approach provides a powerful spatial platform for clinical FFPE specimens and promises enormous applications in biomedicine.
2023-12-31 | GSE247881 | GEO
Project description:Despite the extensive collection of formalin-fixed paraffin-embedded (FFPE) specimens, molecular tools for their in-depth analysis remain limited. Here, we introduce spatial FFPE-ATAC-seq, a technique that enables chromatin accessibility profiling in archival FFPE tissues with spatial resolution. Applied to both human and mouse samples, this method revealed the complex spatial organization and distinct cellular architectures, unlocking the potential of FFPE samples for comprehensive epigenomic studies and human disease research.
2025-05-19 | GSE278007 | GEO
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:Formalin-fixed paraffin-embedded (FFPE) tissues are a vast resource of annotated clinical samples. As such, they represent highly desirable and informative materials for the application of high-definition genomics for improved patient management and to advance the development of personalized therapeutics. However, a limitation of FFPE tissues is the variable quality of DNA extracted for analyses. Furthermore, admixtures of non-tumor and polyclonal neoplastic cell populations limit the number of biopsies that can be studied and make it difficult to define cancer genomes in patient samples. To exploit these valuable tissues, we applied flow cytometry-based methods to isolate pure populations of tumor cell nuclei from FFPE tissues and developed a methodology compatible with oligonucleotide array CGH and whole exome sequencing analyses. These were used to profile a variety of tumors (breast, brain, bladder, ovarian and pancreas), including the genomes and exomes of matching fresh frozen and FFPE pancreatic adenocarcinoma samples.
2012-12-31 | GSE40299 | GEO
Project description:Formalin-fixed paraffin-embedded (FFPE) samples are the gold standard for clinical tissue preservation. However, single-cell chromatin accessibility profiling technology, a powerful tool to understand epigenetic regulation in human disease, is not available for FFPE samples due to extensive DNA damage. To solve this technical challenge, we introduce scFFPE-ATAC by integrating uniquely designed DNA oligos on Tn5 transposase, split-and-pool ligation DNA barcoding, T7 promoter-mediated DNA damage rescue, and in vitro transcription—the first high-throughput single-cell chromatin accessibility assay for FFPE samples. scFFPE-ATAC enables the simultaneous profiling of up to 64 FFPE samples or spatial regions, with 884,736 cell barcodes in a single run. We successfully applied scFFPE-ATAC to clinical FFPE human lymph nodes stored for 8–15 years and identified distinct epigenetic regulators between tumor centers and invasive edge epithelial cells in human lung cancer. Additionally, we analyzed paired primary and relapsed follicular lymphoma (FL), as well as FL transformed into diffuse large B-cell lymphoma, from clinical samples archived for 2–7 years, revealing key epigenetic drivers of tumor progression. Overall, scFFPE-ATAC enables high-throughput, high-sensitivity chromatin accessibility profiling in long-term archived clinical specimens, paving the way for retrospective epigenetic studies and spatial epigenetic analysis. This technology provides a powerful tool for understanding tumor relapse and metastasis, with broad applications in basic research and personalized medicine.
2025-10-14 | GSE291155 | GEO
Project description:This dataset was applied to evaluate the performance of a deep learning framework, FFPERescuer, specifically designed to reconstruct gene expression profiles from RNA sequencing data derived from FFPE (formalin-fixed, paraffin-embedded) tumor tissues. The dataset includes a total of 12 RNA-Seq samples, comprising 10 FFPE tumor tissue samples from colorectal cancer (CRC) cases collected in Amsterdam, the Netherlands, with 2 duplicate samples included for reproducibility assessment. The corresponding gene expression profiles from fresh-frozen tumor tissues for these 10 cases are available in the dataset GSE33113, generated using microarray technology.
2025-07-31 | GSE299799 | GEO
Project description:The proteomics of archival samples has advanced significantly during the last two decades, making possible the use of vast FFPE tissue archives in biomarker studies. However, due to the limited data regarding comparison of the performance of different protocols, there is currently no consensus on the most effective protocol for shotgun proteomic analysis of FFPE tissue. In-solution digestion method using Rapigest as detergent and FASP method, both reported to deliver superior results on FFPE tissues were compared using two label-free data-independent LC-MS/MS acquisition modes. Archival FFPE prostate tissues stored 7 years and mirroring fresh frozen samples were processed with both protocols in 3 technical replicates. Data were comparatively evaluated in terms of protein and peptide identifications in fresh frozen and FFPE samples, analysis of the fresh and FFPE tissues representative proteome (molecular weight distribution, distribution according to protein’s pI, cellular component analysis of proteins and quantitation accuracy) and technical reproducibility of the Rapigest and FASP protocols.
2021-01-15 | PXD021074 | Pride
Project description:Formalin-fixed paraffin-embedded (FFPE) samples are the gold standard for clinical tissue preservation. However, single-cell chromatin accessibility profiling technology, a powerful tool to understand epigenetic regulation in human disease, is not available for FFPE samples due to extensive DNA damage. To solve this technical challenge, we introduce scFFPE-ATAC by integrating uniquely designed DNA oligos on Tn5 transposase, split-and-pool ligation DNA barcoding, T7 promoter-mediated DNA damage rescue, and in vitro transcription—the first high-throughput single-cell chromatin accessibility assay for FFPE samples. scFFPE-ATAC enables the simultaneous profiling of up to 64 FFPE samples or spatial regions, with 884,736 cell barcodes in a single run. We successfully applied scFFPE-ATAC to clinical FFPE human lymph nodes stored for 8–15 years and identified distinct epigenetic regulators between tumor centers and invasive edge epithelial cells in human lung cancer. Additionally, we analyzed paired primary and relapsed follicular lymphoma (FL), as well as FL transformed into diffuse large B-cell lymphoma, from clinical samples archived for 2–7 years, revealing key epigenetic drivers of tumor progression. Overall, scFFPE-ATAC enables high-throughput, high-sensitivity chromatin accessibility profiling in long-term archived clinical specimens, paving the way for retrospective epigenetic studies and spatial epigenetic analysis. This technology provides a powerful tool for understanding tumor relapse and metastasis, with broad applications in basic research and personalized medicine.
2025-10-14 | GSE306401 | GEO
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