Ten percent of aberrant cells is sufficient for detection of DNA copy number alterations
Ontology highlight
ABSTRACT: This SuperSeries is composed of the following subset Series: GSE36856: Ten percent of aberrant cells is sufficient for detection of DNA copy number alterations (part 1) GSE36986: Ten percent of aberrant cells is sufficient for detection of DNA copy number alterations (part 2) GSE36989: Ten percent of aberrant cells is sufficient for detection of DNA copy number alterations (part 3) GSE36991: Ten percent of aberrant cells is sufficient for detection of DNA copy number alterations (part 4) GSE36992: Ten percent of aberrant cells is sufficient for detection of DNA copy number alterations (part 5) Refer to individual Series
Project description:Array Comparative Genomic Hybridization (aCGH) is a widely used technique to assess chromosomal copy number alterations. Chromosomal content, however, is often not uniform throughout cell populations. The aim of our present study is to evaluate to what extent aCGH can detect DNA copy number alterations in a heterogeneous cell population. Reported detection limits are a compound of analytical software and laboratory technique whilst systematic evaluation is lacking, despite the importance in diagnostics and research. Detection limits were explored with DNA isolated from a patient with intellectual disability (ID) and from tumor cell line BT474. Both were diluted with increasing amounts of normal DNA to simulate different levels of cellularity. Samples were hybridized on CGH arrays containing 180880 oligonucleotides evenly distributed over the genome (space ~17kb). The ID sample has a single copy number gain of 4Mb and a single copy number loss of 7.5Mb that could both be detected with 10% mosaicism. The tumor cell line BT474 has a dual copy number gain (6 copies in a background of 4 copies) of 46Mb. This corresponds to a single copy number gain in a diploid sample and could be detected with 15% tumor cells. The diagnostic validity of these findings was verified using two clinical mosaic samples with alterations in 20% (40Mb) and 14% (34Mb) of cells. Both alterations could be accurately detected using t-statistics. In conclusion, single copy number gains and losses, down to 4Mb in as little as 10% of a cell population, can be detected by aCGH. Dilution range of breast cancer cell line BT474 used to test the detection limits of array CGH.
Project description:Array Comparative Genomic Hybridization (aCGH) is a widely used technique to assess chromosomal copy number alterations. Chromosomal content, however, is often not uniform throughout cell populations. The aim of our present study is to evaluate to what extent aCGH can detect DNA copy number alterations in a heterogeneous cell population. Reported detection limits are a compound of analytical software and laboratory technique whilst systematic evaluation is lacking, despite the importance in diagnostics and research. Detection limits were explored with DNA isolated from a patient with intellectual disability (ID) and from tumor cell line BT474. Both were diluted with increasing amounts of normal DNA to simulate different levels of cellularity. Samples were hybridized on CGH arrays containing 180880 oligonucleotides evenly distributed over the genome (space ~17kb). The ID sample has a single copy number gain of 4Mb and a single copy number loss of 7.5Mb that could both be detected with 10% mosaicism. The tumor cell line BT474 has a dual copy number gain (6 copies in a background of 4 copies) of 46Mb. This corresponds to a single copy number gain in a diploid sample and could be detected with 15% tumor cells. The diagnostic validity of these findings was verified using two clinical mosaic samples with alterations in 20% (40Mb) and 14% (34Mb) of cells. Both alterations could be accurately detected using t-statistics. In conclusion, single copy number gains and losses, down to 4Mb in as little as 10% of a cell population, can be detected by aCGH. Dilution range of DNA from a patient with an intellectual disabillity (ID) used to test the detection limits of array CGH.
Project description:To study the oncogenic potential of cyclin D1b in the context of mature B cells we generated several cell clones derived from LP-1 MM cell line expressing either cyclin D1b, Myc or cyclin K oncogenes. Transcriptomic analysis allowed us to describe several mechanisms of cyclin D1b- and K-mediated oncogenesis. Three-condition experiment: LP-1cl1, LP-1K and LP-1D1b. Multiple myeloma LP-1 cell lines. Biological replicates: 4 control (LP-1cl1), 4 transfected with cyclin K (Lp-1K) and 4 transfected with cyclin D1b (LP-1D1b) independently grown and harvested. One replicate per array.
Project description:Transcriptomic profiling of ovine skin lymph dendritic cell subsets CD26+ and CD26- compared to total enriched lymph dendritic cells. CD26+ and CD26- dendritic cells (DC) from sheep skin lymph were sorted by flow cytometry and were analysed for their transcriptomic profile. We demonstrate that the minor sheep CD26+ skin lymph DC subset shares significant transcriptomic similarities with mouse CD8a+ and human BDCA3+ DC. This allowed the identification of a common set of phenotypic characteristics for CD8a-like DC in the 3 mammalian species, i.e. SIRPlo, CADM1hi, CLEC9Ahi, DEC205hi, XCR1hi. Compared to CD26- DC, the sheep CD26+ DC show (1) potent stimulation of allogeneic naive CD8+ T cells with high selective induction of the Ifn-gamma and Il22 genes; (2) dominant efficacy in activating specific CD8+ T cells against exogenous soluble antigen; (3) selective expression of functional pathways associated to high capacity for antigen cross-presentation. Reference design (total enriched lymph dendritic cells) - Biological replicates: 2 sheep #10081 and #30066
Project description:High-grade osteosarcoma is a malignant primary bone tumor with a peak incidence in puberty. Overall survival of patients with metastatic disease is approximately twenty percent. Mechanisms behind the development of metastases in osteosarcoma are unknown. To identify gene signatures that play a role in metastasis, we performed genome-wide gene expression profiling on pre-chemotherapy biopsies of osteosarcoma patients who developed metastases within 5yrs and patients who did not develop metastases within 5yrs. Pre-chemotherapy biopsies of osteosarcoma patients who developed metastases within 5yrs (n=34) were compared with pre-chemotherapy biopsies of osteosarcoma patients who did not develop metastases within 5yrs (n=19).
Project description:Agilent 44K whole genome studies were used to correlate gene expression values with mutations found in massively parallel sequencing experiments. 77 patients were profiled using Agilent's two-color microarray platform. Tumor(cy5) was co-hybridized to a reference(cy3).
Project description:Expression data was used in Paradigm analysis for exploration of networks affected by copy number and gene expression changes based on mutation spectra of recurrently mutated genes in breast cancer. cohort of 124 total patients arrayed and analyzed.
Project description:Transcriptomic profiling of sheep skin lymph DC subsets after 16h in vitro culture in medium or stimulated by non replicative Canine Adenovirus serotype 2 Reference design: total enriched lymph dendritic cells- Biological replicates: 4 sheep (70958, 50274, 30066, 11261)- Samples: FACS sorted CD103 -type and CD11b type cultured in medium or with CAV2
Project description:Screening of 22 novel proteins derived from Campylobacter jejuni NCTC 11168 identified prior via screening of cDNA libraries. The full-length proteins were attached using a specific HaloTag to their corresponding ligand surface, HaloLink. Screening was performed using three different polyclonal antibodies to Campylobacter jejuni and detection was achieved by goat polyclonal antibody to rabbit IgG conjugated with Chromeo-546. In order to assess their potential immungenic nature and rank the proteins investigated, comparative analysis using already described antigens from C. jejuni were used in the assay. Each microarray was separated into different incubation chambers using the ProPlate (Grace Biolabs) multi-well gaskets. While for two slides (2009 and 2447), three chambers were used, the remaining slides were designed to use 16 different compartments. Each compartment could be incubated with different antibodies and represent individual replicates of the slides. As positive references, hisJ and cjaA were used. For negative controls, argC and gapA were used, and the crude lysates of the expression host (Acella E. coli) and buffer were spotted as well. For slides 2009 and 2447, three-well gaskets were used allowing for seven replicates per sample, while only incubation with one antibody. Slides 416033 and 416826 used 16-well gaskets and only hisJ and argC as protein references. Samples and controls were spotted in quadruplicate. Finally, for 1000 and 1001, samples were spotted in triplicate, whereas controls were spotted in quadruplicate using hisJ, cjaA, argC and gapA as protein references.
Project description:Resistance to erythropoietin (EPO) treatment is observed in a considerable proportion of anemic patients with chronic kidney disease. Previous reports suggest that inflammation is one of the major independent predictors of this resistance, and pro-inflammatory cytokines have been shown to inhibit erythropoiesis. The aim of this study was to investigate EPO-induced modification to gene expression in primary cultured leukocytes. Microarray experiments were performed on ex vivo Peripheral Blood Mononuclear Cells (PBMCs) from pooled ten healthy donors (n=9), primed or not with TNF-alpha, and treated with recombinant human erythropoietin alpha (EPO-alpha). Real-time PCR experiments were used to validate expression of the molecular targets considered most relevant for inflammation. Data analysis suggested that EPO-alpha treatment mainly modulated genes involved in cell movement and cell-cell interaction in primed PBMCs. Notably, EPO-alpha exerts anti-inflammatory effects inhibiting the expression of pro-inflammatory cytokine IL-8 and its receptor CXCR2; by contrast, EPO-alpha further increases expression of genes relating to promotion of inflammation encoding for IL-1beta and CCL8, and induces de novo synthesis of IL-1alpha, CXCL1 and CXCL5 in primed PBMCs. MAPK p38alpha?reducing activity is involved in modulation of IL-1beta and IL-8 expression. In conclusion, our findings confirm the anti-inflammatory role for EPO, but also suggest a plausible in vivo scenario, in which the positive correlation found between EPO-resistance and elevated levels of some pro-inflammatory mediators is due to treatment with EPO itself. One future objective will be to perform in vivo measurement of the molecular targets highlighted, in order to identify any additional markers involved in EPO-resistance among hypo-responsive patients. In this study, we analyzed the expression profiles of pooled blood PBMCs from 10 healthy donors primed or not with TNF-alpha and then treated with EPO-alpha. All RNAs from no stimulated PBMCs were hybridized against RNAs from PBMCs treated with TNF-alpha or TNF-alpha and EPO-alpha. We performed a total of 9 technical replicates.