Single Nucleus DNA Sequencing of Flow Sorted Archived Frozen and Formalin Fixed Paraffin Embedded Solid Tumors
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PROVIDER: PRJNA1119467 | ENA |
REPOSITORIES: ENA
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Project description:Archived tissues are a vast resource of annotated clinical samples. To expand their use for translational studies, we optimized flow sorting and sequencing of single nuclei from archived frozen and FFPE tumor tissues. Our methods, which include isolation and preparation of intact nuclei suitable for library preparations, quality control (QC) metrics for each step, and a single cell sequencing bioinformatic processing and analysis pipeline, were validated with flow sorted nuclei from matching frozen and FFPE ovarian cancer surgical samples and a sequencing panel of 553 amplicons targeting single nucleotide and copy number variants in genes of interest.
2024-06-20 | GSE270364 | GEO
Project description:Profiling histone modifications in clinical archived formalin fixed paraffin embedded samples
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Project description:MicroRNAs regulate several aspects of tumorigenesis and cancer progression. Most cancer tissues are archived formalin-fixed and paraffin-embedded (FFPE). While microRNAs are a more stable form of RNA thought to withstand FFPE-processing and degradation there is only limited evidence for the latter assumption. We examined whether microRNA profiling can be successfully conducted on FFPE cancer tissues using SOLiD ligation based sequencing. Tissue storage times (3-9 years) appeared to not affect the number of detected microRNAs in FFPE samples compared to matched frozen samples (paired t-test p>0.7). Correlations of microRNA expression values were very high across microRNAs in a given sample (Pearson’s r=0.71-0.95). Higher variance of expression values among samples was associated with higher correlation coefficients between FFPE and frozen tissues. One of the FFPE samples in this study was degraded for unknown reasons with a peak read length of 17 nucleotides compared to 21 in all other samples. The number of detected microRNAs in this sample was within the range of microRNAs detected in all other samples. Ligation-based microRNA deep sequencing on FFPE cancer tissues is feasible and RNA degradation to the degree observed in our study appears to not affect the number of microRNAs that can be quantified.
2013-04-15 | GSE45740 | GEO
Project description:Tissue sample acquisition is a limiting step in many studies. There are many thousands of formalin fixed paraffin embedded archival blocks collected around the world, but in contrast relatively few fresh frozen samples in tumor banks. Once samples are fixed in formalin the RNA is degraded and traditional methods for gene expression profiling are not suitable. In this study we have evaluated the whole genome DASL assay from Illumina to perform transcriptomic analysis from archived breast tumor tissue fixed in formalin paraffin embedded blocks. We profiled 76 familial breast tumors from cases carrying a BRCA1, BRCA2 or ATM mutation, or from non-BRCA1/2 families. We found that replicate samples correlated well with each other (r2=0.9-0.98). In 12/15 cases, the matched formalin-fixed and frozen samples predicted the same tumor molecular subtypes with confidence. These results demonstrate that the whole genome DASL assay is a valuable tool to profile degraded RNA from archival FFPE material. This assay will enable transcriptomic analysis of a large number of archival samples that are stored in pathology archives around the globe and consequently will have the potential to improve our understanding and characterisation of many diseases.
2010-06-09 | GSE21921 | GEO
Project description:MicroRNAs regulate several aspects of tumorigenesis and cancer progression. Most cancer tissues are archived formalin-fixed and paraffin-embedded (FFPE). While microRNAs are a more stable form of RNA thought to withstand FFPE-processing and degradation there is only limited evidence for the latter assumption. We examined whether microRNA profiling can be successfully conducted on FFPE cancer tissues using SOLiD ligation based sequencing. Tissue storage times (3-9 years) appeared to not affect the number of detected microRNAs in FFPE samples compared to matched frozen samples (paired t-test p>0.7). Correlations of microRNA expression values were very high across microRNAs in a given sample (PearsonM-bM-^@M-^Ys r=0.71-0.95). Higher variance of expression values among samples was associated with higher correlation coefficients between FFPE and frozen tissues. One of the FFPE samples in this study was degraded for unknown reasons with a peak read length of 17 nucleotides compared to 21 in all other samples. The number of detected microRNAs in this sample was within the range of microRNAs detected in all other samples. Ligation-based microRNA deep sequencing on FFPE cancer tissues is feasible and RNA degradation to the degree observed in our study appears to not affect the number of microRNAs that can be quantified. we analyzed eight matched frozen and FFPE tissues of different histologies for which detailed processing and storage information.
2013-04-15 | E-GEOD-45740 | biostudies-arrayexpress
Project description:Formalin-fixed paraffin-embedded (FFPE) ring trial
| PRJEB32144 | ENA
Project description:DNA methylation is the most studied epigenetic modification due to its role in regulating gene expression and aberrations in methylation involved in the pathogenesis of cancer and several diseases. The method of choice to evaluate genome-wide methylation has been the Illumina HumanMethylation450 BeadChip (450K), but it was recently replaced with the MethylationEPIC BeadChip (EPIC). We therefore sought to validate the EPIC array in comparison to the 450K array for both fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) pediatric brain tumors. We also performed analysis on the EPIC array with paired FF and FFPE samples, to adapt to a clinical setting where FFPE is routinely used. Further, we compared two restoration methods, REPLI-g and Infinium, for FFPE-derived DNA on the EPIC array.
2017-04-01 | GSE92579 | GEO
Project description:DNA methylation is the most studied epigenetic modification due to its role in regulating gene expression and aberrations in methylation involved in the pathogenesis of cancer and several diseases. The method of choice to evaluate genome-wide methylation has been the Illumina HumanMethylation450 BeadChip (450K), but it was recently replaced with the MethylationEPIC BeadChip (EPIC). We therefore sought to validate the EPIC array in comparison to the 450K array for both fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) pediatric brain tumors. We also performed analysis on the EPIC array with paired FF and FFPE samples, to adapt to a clinical setting where FFPE is routinely used. Further, we compared two restoration methods, REPLI-g and Infinium, for FFPE-derived DNA on the EPIC array.
2017-04-01 | GSE92577 | GEO
Project description:Background and Aims Formalin-fixed, paraffin-embedded (FFPE) tissue is the most commonly available form of archived clinical specimens, which are often stored as thin sections on glass slides. RNA isolated from such archived section (AS) of FFPE tissue is more degraded compared to freshly cut (FC) FFPE section because of prolonged air exposure. In this study, we evaluated performance of transcriptome profiling-based disease classification in AS-FFPE tissue. Methods Genome-wide gene-expression profiles of AS-FFPE tissues of 83 hepatocellular carcinoma (HCC) and 47 liver cirrhosis samples were generated by using whole-genome DASL assay (Illumina), and compared with the profiles previously produced by using FC tissue sections from the same FFPE blocks. Quality of the profiles and performance of gene signature-based class prediction were systematically evaluated. Results RNA quality and assay reproducibility of AS-FFPE RNA were comparable to intermediate ~ poor quality FC-FFPE samples (R2 as high as 0.93). Gene-expression signal was detected in lower number of probes in AS FFPE samples compared to FC-FFPE samples (proportion of probes with present signal (%P-call): 10%~60% and 70%~90% in AS- and FC-FFPE profiles, respectively). Based on %P-call quality threshold of 20%, 64/88 (77%) HCC and 37/48 (77%) liver profiles were judged as having relatively good quality data with comparable inter-sample correlation. Inter-sample correlation coefficient, as a measure to detect outlier profiles due to poor RNA quality, was also lower in AS-FFPE (0.4~0.9) compared to FC-FFPE (0.6~1.0). In the genome-wide profiling analysis, previously identified molecular subclasses of HCC tumors were reproduced in 67/83 (81%) samples, which was improved to 43/48 (90%) samples when we focused on statistically confident predictions (p<0.05). A 186-gene prognostic signature in liver cirrhosis was reproduced in 32/47 (68%) samples, which was slightly improved to 11/16 (69%) when focused on statistically significant predictions. Conclusions We observed decay of genome-wide transcriptional profiles in AS-FFPE tissues in quantitative manner. However, disease classification was still possible, which suggests potential of AS-FFPE material for clinical diagnosis and prognosis. FFPE tissue sections (10 micron-thick) sliced from 5~16-year-old FFPE blocks and archived for 6~7 years on glass slide Gene-expression profiles of archived section of formalin-fixed paraffin-embedded (AS-FFPE) liver tissues from HCC patients: 47 samples Gene-expression profiles of archived section of formalin-fixed paraffin-embedded (AS-FFPE) tumor tissues from HCC patients: 83 samples
2014-01-31 | E-GEOD-46444 | biostudies-arrayexpress
Project description:In recent years, the use of FFPE tissue in gene expression microarray (GEM) studies has become a topic of increased interest, because pathology departments worldwide contain an invaluable source of biological disease-specific FFPE- tissue material. Thus far, some published studies on FFPE tissue consider the processed tissue amenable, whereas other studies assert that the tissue is not useful in GEM. In general, pancreatic tissue is known to contain large amounts of nucleases, leading to high turnover rates and degradation of RNA in the tissue. This characteristic, in combination with FFPE processing time and its effect on the tissue, does not make FFPE pancreatic tissue the obvious choice for GEM. The basis for using FFPE pancreatic tissue in RNA-based assays seems suboptimal. Previous GEM studies have used different FFPE tissue types, but GEM studies on FFPE pancreatic tissue have not been published. In most studies, the tissue was specially processed for use with GEMs. Here we demonstrate the usefulness of randomly archived FFPE pancreatic tissues for GEMs. For this purpose we included FFPE pancreatic tissue from patients with congenital hyperinsulinism or insulinoma; In these patients, pancreas resection is performed when medical treatment is not adequate to prevent hyperinsulinemic hypoglycemia or when patients do not respond to medical treatment. Although ribonuclease-rich, we obtained biologically relevant and disease-specific, significant genes; cancer-related genes and genes involved in a) the regulation of insulin secretion and synthesis, b) amino acid metabolism, and c) calcium ion homeostasis. This application may extend the possibilities of gene expression studies to many tissue types, especially in rare diseases, for which fresh frozen tissue is not readily available. A total of 15 samples were included in this study using a 58K chip. 29,134 human 60mer oligonucleotide targets were arrayed on the chips in duplicates. Each chip is hybridized with sample material (labelled with Cy5) from one patient and reference material (labelled with Cy3). Reference material contains pooled RNA from four different cancer cell lines; HeLa (cervical epithelium), SK-BR-3 (mammary gland), HT29 (colon) and A431 (skin) cells. Seven patients with congenital hyperinsulinism (five FFPE and two frozen samples), one patient with insulinoma and five controls were included in the study. Inclusion criteria for controls was that their cause of death was not pancreas-related. Samples were obtained from archived FFPE pancreatic material. The insulinoma sample was included in the study, because the biopsy was originally divided with one part being frozen and the other part being routinely formalin-fixed and paraffin-embedded. In the study we therefore include a frozen insulinoma sample and a FFPE insulinoma sample that was processed in duplicate.
2012-09-30 | E-GEOD-32610 | biostudies-arrayexpress