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: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: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:Spatial transcriptomics and multiplexed imaging are complementary methods for studying tissue biology. Here we describe a simple method for transcriptional profiling of formalin fixed histology specimens based on mechanical isolation of tissue micro-regions containing 5-20 cells. Sequencing micro-regions from an archival melanoma specimen having multiple distinct histologies reveals significant differences in transcriptional programs associated with tumor invasion, proliferation, and immunoediting and parallel imaging confirms changes in immuno-phenotypes and cancer cell states.
2021-09-01 | GSE158562 | GEO
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:BackgroundHER2 is considered as one of the most important, predictive biomarkers in oncology. The diagnosis of HER2 positive cancer types such as breast- and gastric cancer is usually based on immunohistochemical HER2 staining of tumour tissue. However, the current immunohistochemical methods do not provide localized information about HER2's functional state. In order to generate signals leading to cell growth and proliferation, the receptor spontaneously forms homodimers, a process that can differ between individual cancer cells.Materials and methodsHER2 overexpressing tumour cells were dissociated from formalin-fixed paraffin-embedded (FFPE) patient's biopsy sections, subjected to a heat-induced antigen retrieval procedure, and immobilized on microchips. HER2 was specifically labelled via a two-step protocol involving the incubation with an Affibody-biotin compound followed by the binding of a streptavidin coated quantum dot (QD) nanoparticle. Cells with membrane bound HER2 were identified using fluorescence microscopy, coated with graphene to preserve their hydrated state, and subsequently examined by scanning transmission electron microscopy (STEM) to obtain the locations at the single molecule level. Label position data was statistically analysed via the pair correlation function, yielding information about the presence of HER2 homodimers.ResultsTumour cells from two biopsies, scored HER2 3+, and a HER2 negative control sample were examined. The specific labelling protocol was first tested for a sectioned tissue sample of HER2-overexpressing tumour. Subsequently, a protocol was optimized to study HER2 homodimerization in single cells dissociated from the tissue section. Electron microscopy data showed membrane bound HER2 in average densities of 201-689 proteins/?m2. An automated, statistical analysis of well over 200,000 of measured protein positions revealed the presence of HER2 homodimers in 33 and 55% of the analysed images for patient 1 and 2, respectively.ConclusionsWe introduced an electron microscopy method capable of measuring the positions of individually labelled HER2 proteins in patient tumour cells from which information about the functional status of the receptor was derived. This method could take HER2 testing a step further by examining HER2 homodimerization directly out of tumour tissue and may become important for adjusting a personalized antibody-based drug therapy.
| S-EPMC6712713 | biostudies-literature
Project description:BackgroundInvestigating the expression of candidate genes in tissue samples usually involves either immunohistochemical labelling of formalin-fixed paraffin-embedded (FFPE) sections or immunofluorescence labelling of cryosections. Although both of these methods provide essential data, both have important limitations as research tools. Consequently, there is a demand in the research community to be able to perform routine, high quality immunofluorescence labelling of FFPE tissues.ResultsWe present here a robust optimised method for high resolution immunofluorescence labelling of FFPE tissues, which involves the combination of antigen retrieval, indirect immunofluorescence and confocal laser scanning microscopy. We demonstrate the utility of this method with examples of immunofluorescence labelling of human kidney, human breast and a tissue microarray of invasive human breast cancers. Finally, we demonstrate that stained slides can be stored in the short term at 4 degrees C or in the longer term at -20 degrees C prior to images being collected. This approach has the potential to unlock a large in vivo database for immunofluorescence investigations and has the major advantages over immunohistochemistry in that it provides higher resolution imaging of antigen localization and the ability to label multiple antigens simultaneously.ConclusionThis method provides a link between the cell biology and pathology communities. For the cell biologist, it will enable them to utilise the vast archive of pathology specimens to advance their in vitro data into in vivo samples, in particular archival material and tissue microarrays. For the pathologist, it will enable them to utilise multiple antibodies on a single section to characterise particular cell populations or to test multiple biomarkers in limited samples and define with greater accuracy cellular heterogeneity in tissue samples.
| S-EPMC2288605 | biostudies-literature
Project description:Aldosterone production is physiologically under the control of circulating potassium and angiotensin II as well as adrenocorticotropic hormone and other secretagogues such as serotonin. The adrenal's capacity to produce aldosterone relies heavily on the expression of a single enzyme, aldosterone synthase (CYP11B2). This enzyme carries out the final reactions in the synthesis of aldosterone and is expressed almost solely in the adrenal zona glomerulosa. From a disease standpoint, primary aldosteronism (PA) is the most common of all adrenal disorders. PA results from renin-independent adrenal expression of CYP11B2 and production of aldosterone. The major causes of PA are adrenal aldosterone-producing adenomas (APA) and adrenal idiopathic hyperaldosteronism. Our understanding of the genetic causes of APA has significantly improved through comprehensive genetic profiling with next-generation sequencing. Whole-exome sequencing has led to the discovery of mutations in six genes that cause renin-independent aldosterone production and thus PA. To facilitate broad-based prospective and retrospective studies of APA, recent technologic advancements have allowed the determination of tumor mutation status using formalin-fixed paraffin-embedded (FFPE) tissue sections. This approach has the advantages of providing ready access to archival samples and allowing CYP11B2 immunohistochemistry-guided capture of the exact tissue responsible for inappropriate aldosterone synthesis. Herein we review the methods and approaches that facilitate the use of adrenal FFPE material for DNA capture, sequencing, and mutation determination.
| S-EPMC8276099 | biostudies-literature
Project description:This SuperSeries is composed of the following subset Series: GSE25231: Expression Profiling of Formalin-Fixed Parafin-Embedded Primary Breast Tumors DASL Cancer Panel GSE25233: Expression Profiling of Formalin-Fixed Parafin-Embedded Primary Breast Tumors Whole Genome DASL Refer to individual Series
| S-ECPF-GEOD-25234 | biostudies-other
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.
| S-ECPF-GEOD-17037 | biostudies-other