Exon array analysis of two human cell lines (HEK293T Ramos B cell)
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Marie-Laure Yaspo
PROVIDER: E-GEOD-13474 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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2012-03-15 | E-GEOD-36017 | biostudies-arrayexpress
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2011-02-19 | E-GEOD-27414 | biostudies-arrayexpress
Project description:BTB and CNC homology 1 (BACH1) is a heme-binding transcription factor repressing the transcription from a subset of MAF recognition elements (MAREs) at low intracellular heme levels. Upon heme binding, BACH1 is released from the MAREs, resulting in increased expression of antioxidant response genes. To systematically address the gene regulatory networks involving BACH1, we performed knock-down of BACH1 in HEK 293T cells using three independent types of small interfering RNAs followed by transcriptome profiling using microarrays. Knockdown of BACH1 in HEK 293T cells was carried out with three different types of siRNA molecules (esiRNA = e, siRNA1 SI00309876 from Qiagen = q and Stealth siRNA2 HSS100910 from Invitrogen = s), followed by RNA extraction after 24h and 72h of knockdown and hybridization on Affymetrix microarrays. For each siRNA type, we performed the experiments in triplicates, with four control replicates (mock transfections without siRNA) at 24h and three control replicates at 72h, resulting in nine samples and four controls at 24h and nine samples and three controls at 72h. Samples at 24h of knockdown: e_1_24, e_2_24, e_3_24, q_1_24, q_2_24, q_3_24, s_1_24, s_2_24, s_3_24. Controls at 24h: m_1_24, m_2_24, m_3_24, m_4_72. Samples at 72h of knockdown: e_1_72, e_2_72, e_3_72, q_1_72, q_2_72, q_3_72, s_1_72, s_2_72, s_3_72. Controls at 72h: m_1_72, m_2_72, m_3_72. REPEATS: biological replicate: BACH1_e_1_24, BACH1_e_2_24, BACH1_e_3_24 biological replicate: BACH1_q_1_24, BACH1_q_2_24, BACH1_q_3_24 biological replicate: BACH1_s_1_24, BACH1_s_2_24, BACH1_s_3_24 biological replicate: BACH1_m_1_24, BACH1_m_2_24, BACH1_m_3_24, BACH1_m_4_24 biological replicate: BACH1_e_1_72, BACH1_e_2_72, BACH1_e_3_72 biological replicate: BACH1_q_1_72, BACH1_q_2_72, BACH1_q_3_72 biological replicate: BACH1_s_1_72, BACH1_s_2_72, BACH1_s_3_72 biological replicate: BACH1_m_1_72, BACH1_m_2_72, BACH1_m_3_72
2011-05-13 | E-GEOD-28050 | biostudies-arrayexpress
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2010-05-14 | E-GEOD-13901 | biostudies-arrayexpress
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2009-11-11 | E-GEOD-13491 | biostudies-arrayexpress
Project description:Data dependent acquisition (DDA) is the method of choice for mass spectrometry based proteomics discovery experiments, data-independent acquisition (DIA) is steadily becoming more important. One of the most important requirement to perform a DIA analysis is the availability of spectral libraries for the peptide identification and quantification. Several researches were already conducted regarding the creation of spectral libraries from DDA analyses and obtaining identifications with these in DIA measurements. But so far only few experiments were conducted, to estimate the effect of these libraries on the quantitative level. In this work we created a spike-in gold standard dataset with known contents and ratios of proteins in a complex sample matrix. With this dataset, we first created spectral libraries using different sample preparation approaches with and without sample prefractionation on peptide and protein level. Two different search engines were used for protein identification. In total, five different spike-in states were compared with DIA analyses, comparing eight different spectral libraries generated by varying approaches and one library free method, as well as one default DDA analysis. Not only the number of identifications on peptide and protein level in the spectral libraries and the corresponding analyses was inspected, but also the number of expected and identified significant quantifications and their ratios were thoroughly examined. We found, that while libraries of prefractionationed samples are generally larger, the actually yielded identifications are not increased compared to repetitive non-fractionated measurements. Furthermore, we show that the accuracy of the quantifications is also highly dependent on the applied spectra library and also whether the peptide or protein level is analysed. Overall, the reproducibility and accuracy of DIA is superior to DDA in all analysed approaches.
2019-11-08 | PXD014956 | Pride
Project description:Data dependent acquisition (DDA) is the method of choice for mass spectrometry based proteomics discovery experiments, data-independent acquisition (DIA) is steadily becoming more important. One of the most important requirement to perform a DIA analysis is the availability of spectral libraries for the peptide identification and quantification. Several researches were already conducted regarding the creation of spectral libraries from DDA analyses and obtaining identifications with these in DIA measurements. But so far only few experiments were conducted, to estimate the effect of these libraries on the quantitative level. In this work we created a spike-in gold standard dataset with known contents and ratios of proteins in a complex sample matrix. With this dataset, we first created spectral libraries using different sample preparation approaches with and without sample prefractionation on peptide and protein level. Two different search engines were used for protein identification. In total, five different spike-in states were compared with DIA analyses, comparing eight different spectral libraries generated by varying approaches and one library free method, as well as one default DDA analysis. Not only the number of identifications on peptide and protein level in the spectral libraries and the corresponding analyses was inspected, but also the number of expected and identified significant quantifications and their ratios were thoroughly examined. We found, that while libraries of prefractionationed samples are generally larger, the actually yielded identifications are not increased compared to repetitive non-fractionated measurements. Furthermore, we show that the accuracy of the quantifications is also highly dependent on the applied spectra library and also whether the peptide or protein level is analysed. Overall, the reproducibility and accuracy of DIA is superior to DDA in all analysed approaches.
2019-11-08 | PXD012986 | Pride
Project description:Background: Transcoelomic spread is the major route of metastasis of ovarian high-grade serous carcinoma (HGSC) with the omentum as the major metastatic site. Its unique tumor microenvironment with its large populations of adipocytes, mesothelial cells and immune cells establishes an intercellular signaling network that is instrumental for metastatic growth yet poorly understood. Methods: Based on transcriptomic analysis of tumor cells, tumor-associated immune and stroma cells we defined intercellular signaling pathways for 284 cytokines and growth factors and their cognate receptors after bioinformatic adjustment for contaminating cell types. The significance of individual components of this network was validated by analyzing clinical correlations and potentially pro-metastatic functions, including tumor cell migration, pro-inflammatory signal transduction and TAM expansion. Results: The data show an unexpected prominent role of host cells, and in particular of omental adipocytes, mesothelial cells and fibroblasts (CAF), in sustaining this signaling network. These cells, rather than tumor cells, are the major source of most cytokines and growth factors in the omental microenvironment (n=176 versus n=13). Many of these factors target tumor cells, are linked to metastasis and are associated with a short survival. Likewise, tumor stroma cells play a major role in both extracellular-matrix-triggered and lipid-mediated signaling. We have verified the functional significance of our observations for three exemplary instances. We show that the omental microenvironment (i) stimulates tumor cell migration via WNT4 which is highly expressed by CAF; (ii) induces pro-tumorigenic TAM proliferation in conjunction with high CSF1 expression by omental stroma cells and (iii) triggers pro-inflammatory signaling, at least in part via a HSP70 – NFkB pathway. Conclusions: The intercellular signaling network of omental metastases is majorly dependent on factors secreted by immune and stroma cells to provide an environment that supports ovarian HGSC progression. Clinically relevant pathways within this network represent novel options for therapeutic intervention.
2021-11-25 | PXD028248 | Pride
Project description:Microenvironment Cell Populations Affymetrix HuGene 1.0ST
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Project description:Electroporation is a versatile method for in vitro or in vivo delivery of different molecules into cells. However, no study so far has analysed the effects of electric pulses used in electrochemotherapy (ECT pulses) or electric pulses used in electrogene therapy (EGT pulses) on malignant cells. We studied the effect of ECT and EGT pulses in human malignant melanoma cells in vitro in order to understand and predict possible effect of electric pulses on gene expression and their possible effect on cell behaviour. We used microarrays with 2698 different oligonucleotides to obtain expression profile of genes involved in apoptosis and development of cancer in a malignant melanoma cell line (SK-MEL28) exposed to ECT pulses and EGT pulses. Cells exposed to ECT pulses showed 68.8% average survival rate and 31.4% average survival rate in cells exposed to EGT pulses. Only seven common genes were found differentially expressed in cells 16 h after exposure to ECT and EGT pulses. We found that ECT and EGT pulses induce HSP70 stress response mechanism, repress histone protein H4, a major protein involved in chromatin assembly, and down-regulate components involved in protein synthesis. Our results show that electroporation does not significantly change expression profile of major tumour suppressor genes or oncogenes of cell cycle. Moreover, electroporation also does not changes the expression of genes involved in stability of DNA, supporting the current evidence that electroporation is a safe method that does not promote tumorigenesis. However, in spite of being considered an isothermal method it does to some extent induce stress that resulted in expression of environmental stress response mechanism, HSP70. The difference in expression of genes involved in development of cancer was obtained by comparison of malignant melanoma cells exposed to EGT or ECT pulses against the same untreated malignant melanoma cells. In our experimental design, microarrays with 2698 different genes were used as a dual colour system in which exposed and non-exposed cellsâ mRNA were separately labelled, mixed and hybridised together on each array. Only microarrays expressing at least 50% of genes were used for further analysis. All oligonucleotides on the same array were spotted in quadruplicates and each microarray analysis was performed in duplicates, therefore obtaining eight measurements of the same oligonucleotide. The acquired data were analysed with Acuity 4.0 to select reliable signals. Only genes, present in both duplicated microarrays were considered for further processing.
2010-06-20 | E-GEOD-15420 | biostudies-arrayexpress