Reconstruction of gene interaction networks by in vitro RNA interference and global expression analysis
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Mark Fellmann
PROVIDER: E-GEOD-12291 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:We here used the technology of RNA interference and cDNA microarrays to reconstruct a network between genes known to be disregulated in ER-positive breast cancer diseases. Therefor single genes of high importance were silenced and the effect of this pertubation was measured with cDNA microarrays Keywords: network_reconstruction_RNAi human ER-positive breast cancer cell line was cultivated as described and transfection with chemically synthesized siRNAs was performed according to standard HiPerFect transfection protocol by Qiagen (Hilden, Germany) At least 4 cDNA-microarrays were hybridized in dye-swap method for the silencing of every gene of interest. To minimize off-target effects we used at least two different siRNAs per gene.
2009-01-28 | E-GEOD-7033 | biostudies-arrayexpress
Project description:End stage heart failure due to ischemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM) have similar characteristics, enlargement of the ventricles, relatively thin-walled ventricle, which leads to a limited contraction force and blood loading. Nevertheless, the response for present therapeutics is very variable and the prognosis is still very bad for ICM and DCM in general. Thus, the ability to differentiate the etiologies of heart failure based structural and physiological changes of the heart would be a step forward to enhance the specificity and the success of given therapy.
2007-10-31 | E-CVDE-1 | biostudies-arrayexpress
Project description:To identify binding partners of Salmonella’s thioredoxin protein encoded by the trxA gene, we performed tandem-affinity purification (TAP) using a C-terminal fusion of thioredoxin with calmodulin-binding peptide and Protein A. TrxA-binding molecules were identified by mass spectrometry (MS) analysis
2023-03-11 | PXD037199 | Pride
Project description:Tumor cell invasion and metastasis are hallmarks of malignancy. Despite recent advances in the understanding of lymphatic spread, the mechanisms by which tumors metastasize to sentinel/distant lymph nodes and beyond are poorly understood. To gain new insights into this complex process, we established a highly metastatic melanoma cell line (B16F1-variant) by in vivo passaging the B16 parental cell line through the lymphatic system. Here, we characterized morphology, rate of cell proliferation, colony formation, migration, tumorogenicity, lymph flow, and capacities to induce tumor- and sentinel lymph node- lymphangiogenesis. Furthermore, microarray-based comparative analysis bewteen parental and passaged cell lines was performed to identify specific gene expression profiles. The most differentially expressed gene was SPP (osteopontin), a secreted glycophosphoprotein which is known to be involved in cancer metastasis. Overexpression of osteopontin in B16 F1-variant was confirmed by Western blot and quantitative RT-PCR. Treatment of cultured lymphatic endothelial cells (LEC) with osteopontin promoted cell migration mediated by the integrin α9 pathway. Our results identify osteopontin as a novel lymphangiogenic factor. B16 and B16 variants were cultured in DMEM supplemented with 20% fetal bovine serum (FBS; Invitrogen, Grand Island, NY) supplemented with antibiotic-antimycotic solution. Every B16 variant was cultured in duplicates. Total cellular RNA was isolated using the Trizol reagent (Invitrogen, Carlsbad, CA) extracted with chloroform, precipitated with isopropanol, washed with 70% ethanol, and dissolved in DNase-free/RNase-free distilled water. The concentration of RNA was measured using NanoDrop ND-1000 spectrophotometer (Witec AG, Littau, Switzerland) and RNA quality was assessed using 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Digoxigenin-UTP–labeled cRNA was generated and amplified from 500ng of total RNA using the NanoAmp RT-IVT Labeling Kit (Applied Biosystems, Foster City, CA) following the manufacturer's protocol, and was hybridized to Applied Biosystems Mouse Genome Survey Microarrays V2.0. Chemiluminescence detection, image acquisition, and analysis were performed using the Chemiluminescence Detection Kit (Applied Biosystems) and the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer following the manufacturer's protocol. A total of two biological replicates were generated for each B16 variant and each cell line data consisted of pooled RNA of two diffferent passage numbers.
2011-11-16 | E-GEOD-33697 | biostudies-arrayexpress
Project description:Cavitation in tuberculosis (TB) enables highly efficient person-to-person aerosol transmission. Currently, a number of hypotheses exist regarding the mechanisms underlying this process. To explore this process, we undertook an unbiased next-generation transcriptional analysis of samples from cavity walls, grossly normal tissue from the same lung, and uninfected control lungs. Of 17,318 mapped host transcripts, only 199 showed a significant >2-fold change in cavity wall versus normal infected and uninfected samples. Amongst 22 upregulated proteases, 5 type I collagenases were over-represented; these included cathepsin K (CTSK), mast cell chymase-1 (CMA1), matrix metalloproteinase (MMP)-1, MMP-13, and MMP-14. To determine if cavitation was associated with changes in collagen turnover, we assayed serum and urinary markers of collagen metabolism in rabbits with cavitary TB versus infected controls. Serum collagen type 1 C-terminal propeptide (CICP, a marker of collagen synthesis and degradation) and urinary deoxypyridinoline (DPD, a marker of degradation) were increased by 10- (p = 0.019) and 4-fold (p = 0.068), respectively; while urinary helical peptide (a marker of degradation) was decreased 3-fold (p=0.015). This indicates that cavitation in TB is associated with significant increases of both destruction and synthesis of collagen and that cleavage of type I collagen occurs outside the known cleavage sites of MMP-1, -13 and -14. Rabbit gene expression analysis in lung tissue from M. tuberculosis infected animals. Cavitary tissue, non cavitary tissue and lung tissue from non-infected animals are compared.
2015-11-03 | E-GEOD-68206 | biostudies-arrayexpress
Project description:TGFBR1*6A is a common hypomorphic variant of the type 1 Transforming Growth Factor Beta Receptor (TGFBR1), which has been associated with increased cancer risk in some studies. Although TGFBR1*6A is capable of switching TGF-β growth inhibitory signals into growth stimulatory signals when stably transfected into MCF-7 breast cancer cells, TGFBR1*6A biological effects are largely unknown. To broadly explore TGFBR1*6A potential oncogenic properties, we assessed its impact on the migration and invasion of MCF-7 cells. We found that TGFBR1*6A significantly enhances MCF-7 cell migration and invasion in a TGF-β signaling independent manner. We set up and performed a gene array using the conditions mimicking the cell migration experiments to determine which genes in the migratory pathway were differentially regulated between the MCF-7*6A cells and the MCF-7*9A (wild type transfected) cells. The gene array identified two downregulated genes in *6A compared to *9A that are involved in cell migration and invasion: ARHGAP5, encoding ARHGAP5, and FN1, encoding fibronectin-1 (FN1). We were subsequently able to use this information in further studies in the lab. Experiment Overall Design: MCF-7 cells were stably transfected with pIRES-TGFBR1-HA-FLAG or pIRES-TGFBR1*6A-HA-FLAG. Clones were picked up and named 6A-SA5 and WT-WB1 referring to the *6A and TGFBR1 clones, respectively. We have found that the *6A mutation causes an increase in migration and invasion of MCF-7 cells which is independent of TGF-beta. We used the gene array to identify genes that were differentially regulated between the two cell lines that could lead to this phenotype. We collected RNA from samples that were serum-deprived overnight prior to being fed with complete media to mimic the conditions in the migration experiment. No exogenous growth factors were added to the media besides the normal 10% heat inactivated FBS. Samples were collected and run in triplicate (referred to in this data set as sample 1, sample 2, and sample 3). The âuntreatedâ refers to the fact that samples were grown in complete media alone with no exogenously added growth factors.
2008-11-22 | E-GEOD-9093 | biostudies-arrayexpress
Project description:We report the genome-wide small RNA of soybean early maturation seed coat parenchyma compartment soybean early maturation seeds using Illumina high-throughput sequencing technology. Illumina sequencing of small RNA from early maturation seed coat parenchyma compartment and early-maturation stage whole seeds
2011-12-22 | E-GEOD-34638 | biostudies-arrayexpress
Project description:During mammalian pre-implantation embryonic development dramatic and orchestrated changes occur in gene transcription. The identification of the complete changes has not been possible until the development of the Next Generation Sequencing Technology. Here we report the first transcriptome dynamics of single matured bovine oocytes and all stages of pre-implantation embryos developed in vivo. Surprisingly, nearly half of the bovine genome, 11,488 to 12,729 genes involved in more than 100 pathways, is expressed in oocytes and early embryos. Despite the similarity in the total numbers of genes expressed across stages, the nature of the expressed genes is dramatically different. A total of 2,845 genes were differentially expressed among different stages, of which the largest change was observed between the 4- and 8-cell stages, demonstrating that the bovine embryonic genome activation occurs at this transition. Additionally, 774 genes were identified as only expressed/highly enriched in particular stages of development. Using weighted gene co-expression network analysis, we found 12 stage-specific modules of co-expressed genes that can be used to represent the corresponding stage of development. Furthermore, we identified conserved key members (or hub genes) of the bovine expressed gene networks. Their vast association with other embryonic genes suggests that they may have important regulatory roles in embryogenesis; yet, the majority of the hub genes are relatively unknown/under-studied in embryos. We also conducted the first embryonic expression profile comparison across three mammalian species, human, mouse and bovine, for which RNA-seq data are available. We found that the three species share more maternally deposited genes than embryonic genome activated genes. More importantly, there are more similarities in embryonic transcriptomes between bovine and humans than between humans and mice, demonstrating that bovine embryos are better models for human embryonic development. This study provides the first comprehensive examination for gene activities in bovine embryos and identified little-known potential master regulators of pre-implantation development. RNA-Seq profiles from single in vivo matured oocytes and in vivo developed embryos from the 2-cell to the blastocyst stages generated using Solid RNA-seq platform.
2014-09-10 | E-GEOD-59186 | biostudies-arrayexpress
Project description:We were able to achieve an initial stable intermediate phase by the transduction of Oct4, Klf4, and c-Myc. Furthermore, over-expression of Sox2 in these intermediate stage cells leads to final iPS cell phase. After examining the gene expression profiles from the initial to final iPS cell phases, we have identified Sox2 downstream genes important for iPS cell induction. 4 groups of cells are analyzed. Each group contains cells from 4 indepent dishes. The four groups are: ES cells, iPS cells, intermediate stage cells (OKM cells) and MEFs
2011-12-29 | E-GEOD-28197 | biostudies-arrayexpress
Project description:Developing effective model systems to evaluate new potential chemotherapeutic reagents is critical. Three-dimensional cell cultures, such as spheroids, aid in bridging the gap between commonly used monolayer cell cultures and significantly more complex and expensive animal models. Spheroid model systems contain pathophysiological and chemical gradients similar to an in vivo tumor, which ultimately form distinct cellular subpopulations. In the spatial SILAC model, labels are pulsed into the spheroid at discrete time windows during development. These media pulses result in labeled proteins in distinct regions of the spheroid, which allows for tracing of quantitative proteomic changes to be correlated to different cellular subregions. In this study, we use the Spatial SILAC model to evaluate the proteomic response of a colon carcinoma spheroid to the multikinase inhibitor Regorafenib. We determined regorafenib to be an effective kinase inhibitor which significantly altered whole spheroid proliferation and resulted in increased apoptosis when the signal was averaged for all cells in the culture. However, when regorafenib treatment is more closely examined among the cellular subpopulations, drastic differences are observed for how the drug impacted the proteome of the necrotic spheroid core and the proliferating outer regions. Whole spheroid and outer region analysis shows that regorafenib treatment inhibited critical pathways such as mTOR signaling, ERK/MAPK signaling, and colorectal cancer metastasis signaling. However, analysis of the core shows that regorafenib had an entirely different effect, including upregulation of MAPK1 and KRAS, possibly indicating drug resistance within these late apoptotic cells. Ultimately, these combinatory studies can be used to further understanding of drug metabolism is different cellular subpopulations and provide valuable information to ultimately improve the accuracy of therapeutic testing.
2024-08-09 | PXD038958 | Pride