High resolution genome wide DNA analysis on a large panel of Human Embryonic stem cell lines
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ABSTRACT: High resolution genome wide DNA analysis on a large panel of Human Embryonic stem cell lines reveals loss of heterozygosity and copy number variation changes associated with culture and affecting gene expression This SuperSeries is composed of the following subset Series: GSE15095: Exon arrays for human embryonic stem cells GSE15096: Affymetrix SNP 6.0 array data for human embryonic stem cell lines
Project description:Samples were used to study if genomic copy number variation changes detected with SNP 6.0 array would correlate with the expression level changes in human embryonic stem cells These samples were used as a portion of the study evaluating the genomic stability and its effects on human embryonic stem cells in prolonged culture. Selected nine human embryonic stem cell samples from different lines having different kind of genomic abnormalities were hybridized to exon arrays.
Project description:Pluripotent human embryonic stem cells are studied for potential applications in regenerative cell replacement therapies because of their untransformed nature and unique capacity to self-renew and differentiate. However it is known that adaptation of hESCs occurs on prolonged culture and may be associated with the acquisition of chromosome abnormalities. We report a high resolution DNA SNP 6.0 array analysis showing copy number variation in 17 different hESC lines maintained in different laboratories. Several of the changes were culture induced and changed the expression levels of affected genes. Loss of heterozygosity (LOH) of chromosome 16q was detected in one line, in addition to several smaller LOH regions. Importantly, hESCs were found to contain several CNVs less than 3 Mb in size which is below the detection limit of conventional karyotyping. Copy number analysis of Affymetrix SNP 6.0 arrays was performed for 29 hESC samples of various passages and laboratory origin. 40 reference HapMap samples from Affymetrix were hybridized at the same time to built a reference genome used in the analysis.
Project description:Samples were prospectively collected from patients with histologically normal surgical resection margins. 96 tissue samples (histologically normal margins, oral carcinoma and adjacent normal tissues) from 24 patients comprised the training set. Our study design was guided by the hypothesis that the expression of genes present in oral squamous cell carcinoma (OSCC) but not in healthy oral tissues would be indicative of recurrence in advance of histological alteration. We used meta-analysis of five published microarray data sets (GEO accession GDS2520, Kuriakose et al. 2004; GDS1584, Toruner et al. 2004; GSE6791, Pyeon et al. 2007; GSE9844, Ye et al. 2008; and GSE10121, Sticht et al. 2008), in conjunction with the current training set, to identify genes reliably over-expressed in OSCC. This reduced gene set was used to train a risk model to predict recurrence based on over-expression of a subset of these genes in histologically normal surgical resection margins. Validation of the risk signature was performed using quantitative real-time reverse-transcription PCR in an independent set of 136 samples from an independent cohort of 30 patients. This was a case-only design involving a training set of 23 tumors and 73 margins from 24 patients with squamous cell carcinoma of the tongue.
Project description:The objective of this study was to examine the effect of the presence of a single or multiple embryo(s) on the transcriptome of the bovine oviduct. In Experiment 1, cyclic (non-bred, n = 6) and pregnant (artificially inseminated, n = 11) heifers were slaughtered on Day 3 after estrus, and the ampulla and isthmic regions of the oviduct ipsilateral to the corpus luteum were separately flushed. Oviductal epithelial cells from the isthmus region, in which all oocytes/embryos were located, were snap-frozen for microarray analysis. In Experiment 2, heifers were divided into cyclic (non-bred, n = 6) or pregnant (multiple embryo transfer, n = 10) groups. In vitro-produced presumptive zygotes were transferred endoscopically to the ipsilateral oviduct on Day 1.5 post estrus (n = 50 zygotes per heifer). Heifers were slaughtered on Day 3 and oviductal isthmus epithelial cells were recovered for RNA sequencing. Microarray analysis in Experiment 1 failed to detect any difference in the transcriptome of the oviductal isthmus induced by the presence of a single embryo. In Experiment 2, following multiple embryo transfer, RNA sequencing revealed 278 differentially expressed genes of which 123 were up- and 155 were down-regulated in pregnant heifers. Most of the down-regulated genes were related to immune function. Five samples from pregnant heifers and five control, cyclic heifers were analysed
Project description:The aim of this study was to compare the transcriptome of the different regions of the oviduct between pregnant and cyclic heifers. After synchronizing crossbred beef heifers, those in standing oestrus (=Day 0) were randomly assigned to cyclic (non bred, n=6), or pregnant (artificially inseminated, n=11) groups. They were slaughtered on Day 3 and both oviducts from each animal were isolated and cut in half to separate ampulla and isthmus. Each portion was flushed to confirm the presence of an oocyte/embryo and was then opened longitudinally and scraped to obtain epithelial cells which were snap-frozen. Oocytes and embryos were located in the isthmus of the oviduct ipsilateral to the corpus luteum. Microarray analysis of oviductal cells revealed that proximity to the corpus luteum did not affect the transcriptome of the isthmus, irrespective of pregnancy status. However, 2287 genes were differentially expressed (P<0.01) between the ampulla and isthmus of the oviduct ipsilateral to the corpus luteum. Gene ontology revealed that the main biological processes overrepresented in the isthmus were synthesis of nitrogen, lipids, nucleotides, steroids and cholesterol as well as vesicle-mediated transport, cell cycle, apoptosis, endocytosis and exocytosis, whereas cell motion, motility and migration, DNA repair, calcium ion homeostasis, carbohydrate biosynthesis and regulation of cilium movement and beat frequency were overrepresented in the ampulla. In conclusion, large differences in gene expression were observed between the isthmus and ampulla that reflect morphological and functional characteristics of each segment. Fifteen samples including 5 pregnant contralateral isthmus heifers, 5 cyclic contralateral isthmus heifers, and 5 pregnant ipsilateral ampulla heifers. Also, ten samples from GSE74593 were reanalyzed with these samples. The full set of processed data are linked at the bottom of the Series record.
Project description:Long noncoding RNAs (lncRNAs) have emerged as important regulators of diverse cellular processes, but their roles in the developing immune system are poorly understood. In this study, we analysed lncRNA expression during human B-cell development by array-based expression profiling of eleven distinct flow-sorted B-cell subsets, comprising pre-B1, pre-B2, immature, naive, memory, and plasma cells from bone marrow biopsies (n=7), and naive, centroblast, centrocyte, memory, and plasmablast cells from tonsil tissue samples (n=6), respectively. A remapping strategy was used to assign the array probes to 37630 gene-level probe sets, reflecting the most recent updates in genomic and transcriptomic databases, which enabled expression profiling of 19579 long noncoding RNAs, comprising 3947 antisense RNAs, 5277 lincRNAs, 7625 pseudogenes, and 2730 additional lncRNAs. As a first step towards inferring the functions of the identified lncRNAs in developing B-cells, we analysed their co-expression with well-characterized protein-coding genes, a method known as â??guilt by associationâ??. By using weighted gene co-expression network analysis, we identified 272 lincRNAs, 471 antisense RNAs, 376 pseudogene RNAs, and 64 lncRNAs within seven sub-networks associated with distinct stages of B-cell development, such as early B-cell development, B-cell proliferation, affinity maturation of antibody, and terminal differentiation. These data provide an important resource for future studies on the functions of lncRNAs in development of the adaptive immune response, and the pathogenesis of B-cell malignancies that originate from distinct B-cell subpopulations. Tonsils were collected from six patients (homo sapiens) during routine tonsillectomy. Mononuclear cells were isolated from tonsils and prepared for multiparametric flow cytometry using an optimized and validated protocol. B-cell subsets of naive, centroblasts, centrocytes, menory and plasmablast cells were isolated and a total of 30 gene expression profiles were generated using the HuEx-1_0-st-v2-micro array chip from Affymetrix.
Project description:Mononuclear cells were isolated from the sternal bone marrow and prepared for multiparametric flow cytometry using an optimized and validated protocol. B-cell subsets of PreBI, PreBII, Immature, Naive, Memory and Plasma cells were isolated and a al of 38 gene expression profiles were generated using the HuEx-1_0-st-v2-micro array chip from Affymetrix to characterize the gene expression in the individual subpopulations. Bone marrow tissue was obtained by physical scraping of the medulla from seven patients (homo sapiens) undergoing cardiac surgery. Mononuclear cells were isolated from bone marrow and prepared for multiparametric flow cytometry using an optimized and validated protocol. B-cell subsets of PreBI, PreBII, Immature, Naive, Memory and Plasma cells were isolated and a total of 38 gene expression profiles were generated using the HuEx-1_0-st-v2-micro array chip from Affymetrix .
Project description:Forced expression of four transcription factors Oct4,Sox2, Klf4 and Myc (OSKM) induces somatic cell reprogramming towards pluripotency. Major efforts have been made to characterize the molecular events involved in this process. Yet, it remains elusive how gene expression change, epigenetic landscape remodelling and cell fate conversion are triggered by expression of these Yamanaka factors.To address this gap,we utilized a secondary inducible reprogramming system and performed genome-wide profilings of Oct4 binding, histone modification(H3K4me3/H3K27me3/H3K4me1/H3K27ac), and gene expression analysis during this process. Through integrative analysis, we revealed stage-specific Oct4 binding and enhancer signatures in consistence with gene expression changes,in which the initial regression of somatic program is followed by the gradual acquisition of pluripotent program. Oct4 preferatially binds to H3K4me1 marked enhancer regions and Oct4 binding is positively correlated with active mark H3K27ac. Moreover,we observed significant enhancer activation of epigenetic related genes, especially acetylation associated genes, prior to pluripotency network activation, suggesting a pivotal role of epigenetic remodelling in the process of pluripotency acquisition and maintenance. We used microarrays to explore the global changes of gene expression during OSKM-mediated somatic cell reprogramming. time series design with bulk population samples collected at different time point of 2nd reprogramming as well as a corresponding iPS cell line. Each sample include two replicates. 2nd mouse embryonic fibroblasts(sample day0) were treated with ES medium supplemented with 1µg/ml Doxcycline and 50µg/ml Vitamin C for 15 days (sample day1-day15); 72 hours after dox and Vc withdrawl, dox-independent iPS cells are collected(sample day18).
Project description:Germline and somatic mutations in BRCA1predispose to breast cancer. We found that proteasome inhibitors can selectively kill BRCA1-depleted cells. The toxic response involves a deregulation of the G1/S cell cycle checkpoint via hyperphosphorylation of RB1, 53BP1-mediated arrest at G2/M checkpoint, and ERN1-mediated unfolded protein response, culminating in a TNF receptor-mediated apoptosis. The study new unexpected molecular functions for BRCA1 protein and opens a novel possibility for the treatment of BRCA1-deficient cancers. We used microarrays to detail the global programme of gene expression underlying the response of BRCA1-deficient cells to proteasome inhibitor bortezomib. We aimed to identify genes that are strongly up- or down-regulated with a combination of BRCA1 knockdown and proteasome inhibition, but none of these treatments alone before the onset of apoptosis. HeLa and U2OS cells were transfected either with a non-targeting or anti-BRCA1 siRNAs (siControl or siBRCA1, respectively), treated with bortezomib for 8 hours, after which RNA was extracted for hybridization on Affymetrix microarray. The following treatments have been performed: (T1) siControl; (T2) siControl + 20 nM bortezomib for 8h; (T3) siBRCA1; (T4) siBRCA1 + 20 nM bortezomib for 8h. All samples were used without replicas. However, all genes showing inconsistent expression pattern between the two cell lines were excluded from further consideration. Selected candidate genes were subject to validation by qRT-PCR.