Whole-Genome Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded Tissue Samples
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ABSTRACT:
OTHER RELATED OMICS DATASETS IN: PRJNA123271PRJNA123269PRJNA123359PRJNA118511PRJNA123357PRJNA123347PRJNA123361PRJNA123349PRJNA123351
ORGANISM(S): Homo sapiens
PROVIDER: GSE17599 | GEO | 2010/01/13
SECONDARY ACCESSION(S): PRJNA118511
REPOSITORIES: GEO
Similar Datasets
Project description:BackgroundWe have developed a gene expression assay (Whole-Genome DASL), capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE) specimens.Methodology/principal findingsWe demonstrated a similar level of sensitivity in gene detection between matched fresh-frozen (FF) and FFPE samples, with the number and overlap of probes detected in the FFPE samples being approximately 88% and 95% of that in the corresponding FF samples, respectively; 74% of the differentially expressed probes overlapped between the FF and FFPE pairs. The WG-DASL assay is also able to detect 1.3-1.5 and 1.5-2 -fold changes in intact and FFPE samples, respectively. The dynamic range for the assay is approximately 3 logs. Comparing the WG-DASL assay with an in vitro transcription-based labeling method yielded fold-change correlations of R(2) approximately 0.83, while fold-change comparisons with quantitative RT-PCR assays yielded R(2) approximately 0.86 and R(2) approximately 0.55 for intact and FFPE samples, respectively. Additionally, the WG-DASL assay yielded high self-correlations (R(2)>0.98) with low intact RNA inputs ranging from 1 ng to 100 ng; reproducible expression profiles were also obtained with 250 pg total RNA (R(2) approximately 0.92), with approximately 71% of the probes detected in 100 ng total RNA also detected at the 250 pg level. When FFPE samples were assayed, 1 ng total RNA yielded self-correlations of R(2) approximately 0.80, while still maintaining a correlation of R(2) approximately 0.75 with standard FFPE inputs (200 ng).Conclusions/significanceTaken together, these results show that WG-DASL assay provides a reliable platform for genome-wide expression profiling in archived materials. It also possesses utility within clinical settings where only limited quantities of samples may be available (e.g. microdissected material) or when minimally invasive procedures are performed (e.g. biopsied specimens).
| S-EPMC2780295 | biostudies-literature
Project description:A substantial proportion of tumors consist of genotypically distinct subpopulations of cancer cells. This intratumor genetic heterogeneity poses a substantial challenge for the implementation of precision medicine. Single-cell genomics constitutes a powerful approach to resolve complex mixtures of cancer cells by tracing cell lineages and discovering cryptic genetic variations that would otherwise be obscured in tumor bulk analyses. Because of the chemical alterations that result from formalin fixation, single-cell genomic approaches have largely remained limited to fresh or rapidly frozen specimens. Here we describe the development and validation of a robust and accurate methodology to perform whole-genome copy-number profiling of single nuclei obtained from formalin-fixed paraffin-embedded clinical tumor samples. We applied the single-cell sequencing approach described here to study the progression from in situ to invasive breast cancer, which revealed that ductal carcinomas in situ show intratumor genetic heterogeneity at diagnosis and that these lesions may progress to invasive breast cancer through a variety of evolutionary processes.
| S-EPMC5608257 | biostudies-literature
Project description:Archived formalin-fixed paraffin-embedded (FFPE) samples are the global standard format for preservation of the majority of biopsies in both basic research and translational cancer studies, and profiling chromatin accessibility in the archived FFPE tissues is fundamental to understanding gene regulation. Accurate mapping of chromatin accessibility from FFPE specimens is challenging because of the high degree of DNA damage. Here, we first showed that standard ATAC-seq can be applied to purified FFPE nuclei but yields lower library complexity and a smaller proportion of long DNA fragments. We then present FFPE-ATAC, the first highly sensitive method for decoding chromatin accessibility in FFPE tissues that combines Tn5-mediated transposition and T7 in vitro transcription. The FFPE-ATAC generates high-quality chromatin accessibility profiles with 500 nuclei from a single FFPE tissue section, enables the dissection of chromatin profiles from the regions of interest with the aid of hematoxylin and eosin (H&E) staining, and reveals disease-associated chromatin regulation from the human colorectal cancer FFPE tissue archived for >10 yr. In summary, the approach allows decoding of the chromatin states that regulate gene expression in archival FFPE tissues, thereby permitting investigators to better understand epigenetic regulation in cancer and precision medicine.
| S-EPMC8744681 | biostudies-literature
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.
| S-EPMC3511535 | biostudies-literature
Project description:Transcriptome profiling can provide information of great value in clinical decision-making, yet RNA from readily available formalin-fixed paraffin-embedded (FFPE) tissue is often too degraded for quality sequencing. To assess the clinical utility of FFPE-derived RNA, we performed ribo-deplete RNA extractions on?>?3200 FFPE slide samples; 25 of these had direct FFPE vs. fresh frozen (FF) replicates, 57 were sequenced in 2 different labs, 87 underwent multiple library analyses, and 16 had direct microdissected vs. macrodissected replicates. Poly-A versus ribo-depletion RNA extraction methods were compared using transcriptomes of TCGA cohort and 3116 FFPE samples. Compared to FF, FFPE transcripts coding for nuclear/cytoplasmic proteins involved in DNA packaging, replication, and protein synthesis were detected at lower rates and zinc finger family transcripts were of poorer quality. The greatest difference in extraction methods was in histone transcripts which typically lack poly-A tails. Encouragingly, the overall sequencing success rate was 81%. Exome coverage was highly concordant in direct FFPE and FF replicates, with 98% agreement in coding exon coverage and a median correlation of whole transcriptome profiles of 0.95. We provide strong rationale for clinical use of FFPE-derived RNA based on the robustness, reproducibility, and consistency of whole transcriptome profiling.
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Project description:Performance assayed with FFPE tissues
2010-01-13 | GSE17572 | GEO
Project description:To date, few studies have systematically characterized microarray gene expression signal performance with degraded RNA from fixed (FFPE) in comparison with intact RNA from unfixed fresh-frozen (FF) specimens. RNA was extracted and isolated from paired tumor and normal samples from both FFPE and FF kidney, lung, and colon tissue specimens and microarray signal dynamics on both the raw probe and probeset level were evaluated. A contrast metric was developed to directly compare microarray signal derived from RNA extracted from matched FFPE and FF specimens. Gene-level summaries were then compared to determine the degree of overlap in expression profiles. RNA extracted from FFPE material was more degraded and fragmented than FF, resulting in a reduced dynamic range of expression signal. In addition, probe performance was not affected uniformly and declined sharply toward 5' end of genes. The most significant differences in FFPE versus FF signal were consistent across three tissue types and enriched with ribosomal genes. Our results show that archived FFPE samples can be used to profile for expression signatures and assess differential expression similar to unfixed tissue sources. This study provides guidelines for application of these methods in the discovery, validation, and clinical application of microarray expression profiling with FFPE material.
| S-EPMC2893624 | biostudies-literature
Project description:Assay performance with low RNA inputs
2010-01-13 | GSE17565 | GEO
Project description:Formalin fixing with paraffin embedding (FFPE) has been a standard sample preparation method for decades, and archival FFPE samples are still very useful resources. Nonetheless, the use of FFPE samples in cancer genome analysis using next-generation sequencing, which is a powerful technique for the identification of genomic alterations at the nucleotide level, has been challenging due to poor DNA quality and artificial sequence alterations. In this study, we performed whole-exome sequencing of matched frozen samples and FFPE samples of tissues from 4 cancer patients and compared the next-generation sequencing data obtained from these samples. The major differences between data obtained from the 2 types of sample were the shorter insert size and artificial base alterations in the FFPE samples. A high proportion of short inserts in the FFPE samples resulted in overlapping paired reads, which could lead to overestimation of certain variants; >20% of the inserts in the FFPE samples were double sequenced. A large number of soft clipped reads was found in the sequencing data of the FFPE samples, and about 30% of total bases were soft clipped. The artificial base alterations, C>T and G>A, were observed in FFPE samples only, and the alteration rate ranged from 200 to 1,200 per 1M bases when sequencing errors were removed. Although high-confidence mutation calls in the FFPE samples were compatible to that in the frozen samples, caution should be exercised in terms of the artifacts, especially for low-confidence calls. Despite the clearly observed artifacts, archival FFPE samples can be a good resource for discovery or validation of biomarkers in cancer research based on whole-exome sequencing.
| S-EPMC4671711 | biostudies-literature
Project description:Endometriosis is an inflammatory pathology associated with a negative effect on life quality. Recently, this pathology was connected to ovarian cancer, in particular with endometrioid ovarian cancer. microRNAs (miRNAs) are a class of RNA transcripts ~19-22 nucleotides in length, the altered miRNA pattern being connected to pathological status. miRNAs are highly stable transcripts, and these can be assessed from formalin-fixed paraffin-embedded (FFPE) samples leading to the identification of miRNAs that could be developed as diagnostic and prognostic biomarkers, in particular those involved in malignant transformation. The aim of our study was to evaluate miRNA expression pattern in FFPE samples from endometriosis and ovarian cancer patients using PCR-array technology and also to compare the differential expression pattern in ovarian cancer versus endometriosis. For the PCR-array study, we have used nine macrodissected FFPE samples from endometriosis tissue, eight samples of ovarian cancers and five normal ovarian tissues. Quantitative real-time PCR (qRT-PCR) was used for data validation in a new patient cohort of 17 normal samples, 33 endometriosis samples and 28 ovarian cancer macrodissected FFPE samples. Considering 1.5-fold expression difference as a cut-off level and a P-value <0.05, we have identified four miRNAs being overexpressed in endometrial tissue, while in ovarian cancer 15 were differentially expressed (nine overexpressed and six downregulated). The expression level was confirmed by qRT-PCR for miR-93, miR-141, miR-155, miR-429, miR-200c, miR-205 and miR-492. Using the interpretative program Ingenuity Pathway Analysis revealed several deregulated pathways due to abnormal miRNA expression in endometriosis and ovarian cancer, which in turn is responsible for pathogenesis; this differential expression of miRNAs can be exploited as a therapeutic target. A higher number of altered miRNAs were detected in endometriosis versus ovarian cancer tissue, most of them being linked with epithelial-to-mesenchymal transition.
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