RNA-sequencing of formalin fixed human primary melanoma tissue
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE126076 | GEO | 2019/02/05
REPOSITORIES: GEO
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Project description:RNA-sequencing of formalin fixed human primary melanoma tissue
| PRJNA520916 | ENA
Project description:Next Generation Sequencing (NGS) technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined. We conducted a study to determine whether DNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries Residual Tissue Repositories (RTR) was present in sufficient quantity and quality for NGS assays. Fifty-nine FFPE tissues, stored from 3 to 32 years, were obtained from three SEER RTR sites. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing (WES). Following DNA extraction, 58 of 59 specimens (98%) yielded DNA and moved on to the library generation step followed by WES. Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining. Overall, 90% (53/59) of specimens provided usable NGS data regardless of storage time. This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS.
| S-EPMC4519244 | biostudies-literature
Project description:miRNA expression analysis was done on Uveal melanoma, metastatic and non - metastatic case in formalin fixed paraffin embedded sections, identified from case registry and confirmed by Chromosome insitu hybridization harboring monosomy 3 aberration in metastaic case and no aberration in non metastatic case. 19 miRNAs were found to be expressed only in class I tumors (choroidal melanoma) and not in class II (liver metastasis) and 11 miRNAs were found to be expressed only in class II and not in class I. The tumors were found to harbor oncomirs in both choroidal melanoma and metastasizing melanoma targeting tumor suppressor genes and metastatic suppressor genes. None of the differentially expressed miRNAs in either case were found to be located on the chromosomes which have been proved to carry chromosomal abnormalities. Rather it was found that genes targeted by the miRNAs were found to be present in chromosomal regions that are often found to be deleted 8p22, 13q and 17p.
2009-07-11 | GSE17037 | GEO
Project description:Initiatives such as The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) have generated high-quality, multi-platform molecular data from thousands of frozen tumor samples. While these initiatives have provided invaluable insight into cancer biology, a tremendous potential resource remains largely untapped in formalin-fixed, paraffin-embedded (FFPE) samples that are more readily available, but which can present technical challenges due to crosslinking of fragile molecules such as RNA. We extracted RNA from FFPE primary melanomas and assessed two gene expression platforms -- genome-wide RNA sequencing (RNA-seq) and targeted NanoString -- for their ability to generate coherent biological signals. To do so, we generated an improved approach to quantifying gene expression pathways, in which we refine pathway scores through correlation-guided gene subsetting. We also make comparisons to the TCGA and other publicly available melanoma datasets. Comparison of the gene expression patterns to each other, to established biological modules, and to clinical and immunohistochemical data confirmed the fidelity of biological signals from both platforms using FFPE samples to known biology. Moreover, correlations with patient outcome data were consistent with previous frozen-tissue-based studies. FFPE samples from previously difficult-to-access cancer types - such as small primary melanomas - represents a valuable and previously unexploited source of analyte for RNA-seq and NanoString platforms. This work provides an important step towards the use of such platforms to unlock novel molecular underpinnings and inform future biologically-driven clinical decisions.
| S-EPMC6498859 | biostudies-literature
Project description:miRNA expression analysis was done on Uveal melanoma, metastatic and non - metastatic case in formalin fixed paraffin embedded sections, identified from case registry and confirmed by Chromosome insitu hybridization harboring monosomy 3 aberration in metastaic case and no aberration in non metastatic case. 19 miRNAs were found to be expressed only in class I tumors (choroidal melanoma) and not in class II (liver metastasis) and 11 miRNAs were found to be expressed only in class II and not in class I. The tumors were found to harbor oncomirs in both choroidal melanoma and metastasizing melanoma targeting tumor suppressor genes and metastatic suppressor genes. None of the differentially expressed miRNAs in either case were found to be located on the chromosomes which have been proved to carry chromosomal abnormalities. Rather it was found that genes targeted by the miRNAs were found to be present in chromosomal regions that are often found to be deleted 8p22, 13q and 17p. Organism used: Homo sapiens. * Slides: Human miRNA V2 8x15k Arrays AMADID: 19118. * Starting material: 18 slides with formalin fixed Paraffin embedded sections-20 μm. * RNA Samples used: 225-94-DUP, 225-94-E, 727-07-DUP, 727-07 -E. * Labeling kit: Agilent miRNA labeling reagent and Hybridization Kit. * Labeling Method: Ligation of one cyanine3-pCp molecule to the 3' end of RNA molecule with greater than 90% efficiency that generates fluorescent miRNA. * Total RNA and cRNA Purification Kit: Biorad MicroBio Spin 6 columns. * Hybridization Kit: Agilent miRNA Hybridization kit. Hybridization protocol See Agilent Technologies website for miRNA microarray hybridization and wash protocol: http://www.chem.agilent.com/scripts/LiteraturePDF.asp?iWHID=50500 Scan protocol Laser detection of Cyanine 3 and Cyanine 5 fluorescence is performed using a confocal scanning instrument containing two tuned lasers, which excite Cyanine dyes at the appropriate wavelengths. Description Microarrays were scanned on an Agilent scanner (G2505C) at 100% PMT with 10% for lower intensity (XDR Scanning).Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturer’s protocol Data processing Feature extracted data was analyzed using GeneSpring GX v 10.0.2 software from Agilent. Normalization of the data was done in GeneSpring GX using the recommended Percentile shift normalization (50th percentile) . Further quality control of normalized data was done using correlation based condition tree to eliminate bad experiments. High expressed miRNA's were clustered using gene tree to identify significant gene expression patterns.
2009-07-11 | E-GEOD-17037 | biostudies-arrayexpress
Project description:The use of fresh tissue for molecular studies is preferred but often impossible. Instead, frozen or formalin-fixed, paraffin-embedded (FFPE) tissues are widely used and constitute valuable resources for retrospective studies. We assessed the utility of cardiac tissue stored in different ways for gene expression analyses by whole transcriptome sequencing of paired fresh, frozen, and FFPE tissues. RNA extracted from FFPE was highly degraded. Sequencing of RNA from FFPE tissues yielded higher proportions of intronic and intergenic reads compared to RNA from fresh and frozen tissues. The global gene expression profiles varied with the storage conditions, particularly mitochondrial and long non-coding RNAs. However, we observed high correlations among protein-coding transcripts (ρ > 0.94) with the various storage conditions. We did not observe any significant storage effect on the allele-specific gene expression. However, FFPE had statistically significantly (p < 0.05) more discordant variant calls compared to fresh and frozen tissue. In conclusion, we found that frozen and FFPE tissues can be used for reliable gene expression analyses, provided that proper quality control is performed and caution regarding the technical variability is withheld.
| S-EPMC10058139 | biostudies-literature
Project description:Current genomic studies are limited by the poor availability of fresh-frozen tissue samples. Although formalin-fixed diagnostic samples are in abundance, they are seldom used in current genomic studies because of the concern of formalin-fixation artifacts. Better characterization of these artifacts will allow the use of archived clinical specimens in translational and clinical research studies. To provide a systematic analysis of formalin-fixation artifacts on Illumina sequencing, we generated 26 DNA sequencing data sets from 13 pairs of matched formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FF) tissue samples. The results indicate high rate of concordant calls between matched FF/FFPE pairs at reference and variant positions in three commonly used sequencing approaches (whole genome, whole exome, and targeted exon sequencing). Global mismatch rates and C · G > T · A substitutions were comparable between matched FF/FFPE samples, and discordant rates were low (<0.26%) in all samples. Finally, low-pass whole genome sequencing produces similar pattern of copy number alterations between FF/FFPE pairs. The results from our studies suggest the potential use of diagnostic FFPE samples for cancer genomic studies to characterize and catalog variations in cancer genomes.
| S-EPMC4694877 | biostudies-literature
Project description:The dominant genetic signature found in melanoma is C to T mutations due to UV radiation. Nucleotide excision repair (NER) recognises and repairs UV-induced DNA damage. We aimed to determine the effect of high UV exposure on the melanoma transcriptome and key NER transcripts in melanoma tumours in association with clinical and genetic features of the disease. 196 primary or metastatic melanomas were utilised for this study. Solar elastosis and transcriptome data was collected. mRNA transcript levels of NER components XPC, DDB1 and DDB2 were quantified and compared to clinical parameters. Solar elastosis negatively correlated with Breslow thickness (-0.251, p=0.017) and was significantly lower in BRAFV600E melanomas. Lower XPC expression was associated with BRAFV600E and NRASQ61R mutations, earlier age of diagnosis and poorer survival. Transcriptome profiling identified an over-representation of DNA repair processes in high vs low solar elastosis. Further analysis revealed DNA repair and kinase activity related transcripts in the low XPC melanomas. XPC deficiency is associated with the presence of kinase mutations and an aggressive disease phenotype, both of which result from the hypermutability of melanoma.
2016-12-31 | GSE59455 | GEO
Project description:Accurate transcriptional sequencing (RNA-seq) from formalin-fixation and paraffin-embedding (FFPE) tumor samples presents an important challenge for translational research and diagnostic development. In addition, there are now several different protocols to prepare a sequencing library from total RNA. We evaluated the accuracy of RNA-seq data generated from FFPE samples in terms of expression profiling.We designed a biospecimen study to directly compare gene expression results from different protocols to prepare libraries for RNA-seq from human breast cancer tissues, with randomization to fresh-frozen (FF) or FFPE conditions. The protocols were compared using multiple computational methods to assess alignment of reads to reference genome, and the uniformity and continuity of coverage; as well as the variance and correlation, of overall gene expression and patterns of measuring coding sequence, phenotypic patterns of gene expression, and measurements from representative multigene signatures.The principal determinant of variance in gene expression was use of exon capture probes, followed by the conditions of preservation (FF versus FFPE), and phenotypic differences between breast cancers. One protocol, with RNase H-based rRNA depletion, exhibited least variability of gene expression measurements, strongest correlation between FF and FFPE samples, and was generally representative of the transcriptome from standard FF RNA-seq protocols.Method of RNA-seq library preparation from FFPE samples had marked effect on the accuracy of gene expression measurement compared to matched FF samples. Nevertheless, some protocols produced highly concordant expression data from FFPE RNA-seq data, compared to RNA-seq results from matched frozen samples.
| S-EPMC5976456 | biostudies-other
Project description:Adequate access to fresh or frozen normal adrenal tissue has been a primary limitation to the enhanced characterization of the adrenal zones via RNA sequencing (RNAseq). Herein, we describe the application of targeted RNAseq to formalin-fixed paraffin-embedded (FFPE) normal adrenal gland specimens. Immunohistochemistry (IHC) was used to visualize and guide the capture of the adrenocortical zones and medulla. Following IHC-based tissue capture and isolation of RNA, high-throughput targeted RNAseq highlighted clear transcriptomic differences and identified differentially expressed genes among the adrenal zones. Our data demonstrate the ability to capture FFPE adrenal zone tissue for targeted transcriptomic analyses. Future comparison of normal adrenal zones will improve our understanding of transcriptomic patterns and help identify potential novel pathways controlling zone-specific steroid production.
| S-EPMC8456741 | biostudies-literature