The goal of this study is to compare gene expression profiles in quiescent RPE1 hTert cells treated with microtubule-stabilizing (paclitaxel) and microtubule-destabilizing poisons (combretastatin A4) Overall design: RPE 1 hTert cells were grown to full confluency, and maintained as such for 5 days to induce quiescence. Quiescent cells were treated with microtubule poisons combretastatin A4 and paclitaxel for 6 or 24 hours. Total RNA was collected and purified using the PureLink RNA Mini Kit (Invitrogen, Thermo Fisher Scientific, USA). RNA concentration and quality were determined using NanoDrop and Bioanalyzer respectively, and 500 ng of purified RNA was used as input for the Illumina TruSeq Stranded mRNA Library Prep Kit (Illumina, USA). Barcoded libraries were pooled and quantitated using KAPA, and single-end sequenced on an Illumina NextSeq (Illumina, USA). RNA-seq reads were mapped using STAR (version 2.1.0j) and processed using HTSeq-count (version 0.6.1). GRCh38 reference genome and transcript annotations were used for gene mapping; Entrez Gene identifiers and org.Hs.eg.db database were used for genome wide annotation. Differential gene expression and statistical analysis were performed using edgeR package. Genes with >50 reads per million and a fold change significantly different from zero in Wilcoxon signed-rank test (p< 0.05), were marked as differentially expressed genes, based on three biological replicates.
Tubulin mRNA stability is sensitive to change in microtubule dynamics caused by multiple physiological and toxic cues.
Specimen part, Subject
View SamplesAs part of a study of the role of the aryl hydrocarbon receptor (Ahr) in maintenance and senescence of hematopoietic stem cells (HSC), global gene expression profiling was done with HSC isolated from 18-month-old Ahr-knockout and wild-type mice. HSC from aged AhR-KO mice had changes in expression of many genes related to HSC maintenance, consistent with the phenotype observed in aging Ahr-KO mice: decreased survival rate, splenomegaly, increased circulating white blood cells, hematopoietic cell accumulation in tissues, anemia, increased numbers of stem/progenitor and lineage-committed cells in bone marrow, decreased erythroid progenitor cells in bone marrow, and decreased self-renewal capacity of HSC.
Conditional deletion of Ahr alters gene expression profiles in hematopoietic stem cells.
No sample metadata fields
View SamplesAs part of a study of the role of the aryl hydrocarbon receptor (Ahr) in maintenance and senescence of hematopoietic stem cells (HSC), global gene expression profiling was done with HSC isolated from Ahr-knockout and wild-type mice. HSC from young-adult (8 wk old) AhR-KO mice had changes in expression of many genes related to HSC maintenance, consistent with the phenotype observed in aging Ahr-KO mice: decreased survival rate, splenomegaly, increased circulating white blood cells, hematopoietic cell accumulation in tissues, anemia, increased numbers of stem/progenitor and lineage-committed cells in bone marrow, decreased erythroid progenitor cells in bone marrow, and decreased self-renewal capacity of HSC.
Loss of aryl hydrocarbon receptor promotes gene changes associated with premature hematopoietic stem cell exhaustion and development of a myeloproliferative disorder in aging mice.
Specimen part
View SamplesThis experiment was designed to identify genes expressed preferentially in the two integuments of the Arabidopsis ovule. Pistils from wild type and two ovule mutants were compared against each aintegumenta-4 (ant-4) which lacks both integuments and inner no outer (ino-1) which lacks the outer integument. Genes that are highly expressed only in the integuments were expected to be reduced in expression in the mutants, as compared with wild type. Pistils containing ovules through all stages of ovule development prior to pollination were pooled for one experiment (FULL arrays), and for two separate experiments, a set of early differentiation stages (EARLY arrays) and a set of later differentiation stages (LATE ARRAYS) were pooled. Wild type and mutant lines are in the ecotype Landsberg erecta.
Expression-based discovery of candidate ovule development regulators through transcriptional profiling of ovule mutants.
Specimen part
View SamplesWe used RNA-Seq to compare transcriptomes of chemical reprogramming competent worms versus worms not competent for chemical reprogramming. We also performed RNA-seq during a time course of chemical reprogramming. Overall design: Three replicates of each of two reprogramming non-competent strains and three replicates of each of two reprogramming competent strains were collected. For the time course, five time points were analyzed (1, 2, 4, 6, and 18 hours) in either DMSO or DMSO + U0126 in three genotypes (non-reprogramming competent worms, reprogramming competent, and wildtype worms).
Competence for chemical reprogramming of sexual fate correlates with an intersexual molecular signature in Caenorhabditis elegans.
Subject
View SamplesThe basic helix-loop-helix (bHLH) transcription factors of the Drosophilas atonal-related superfamily Neurogenin3 (Neurog3) and NeuroD1 promote endocrine differentiation in the gastrointestinal tract. Atonal Homolog 8 (Atoh8/Math6) is a newly identified member of the atonal-related family whose expression is induced by Neurog3 and NeuroD1 in cell culture, indicating a possible role for this gene in the endocrine differentiation program downstream of these two pro-endocrine factors. Intriguingly, available experimental evidence based on a reduced number of genes suggests that Atoh8 may negatively regulate Neurog3-targeting events. In this study, we have analyzed global changes in gene expression profiles upon exogenous expression of Atoh8 alone or in combination with Neurog3 in mouse pancreatic duct (mPAC) cells. These cells activate neuroendocrine-specific gene expression in response to Neurog3 and NeuroD1 and thus serve as an optimal model to evaluate the proendocrine activity of Atoh8. We have compared transcriptional profiles between mPAC cells treated with a recombinant adenovirus expressing Atoh8 (Ad-Atoh8) or a control adenovirus encoding B-galactosidase (Ad-Bgal), and between cells treated with Ad-Neurog3+Ad-Bgal or cells treated with Ad-Neurog3+Ad-Atoh8. The results obtained show that Atoh8 exhibits a very modest transcriptional activity in these cells thus confirming that Atoh8 does not function as a proendocrine gene. Furthermore, our data also confirm the ability of Atoh8 to block Neurog3-dependent transcriptional activation events. However, since repression is only seen for a small subset of Neurog3 gene targets, we discard a general role of Atoh8 as a negative regulator of Neurog3 pro-endocrine activity.
Characterization of the transcriptional activity of the basic helix-loop-helix (bHLH) transcription factor Atoh8.
Cell line, Treatment
View SamplesMicroarray expression profiling has become a valuable tool in the evaluation of the genetic consequences of metabolic disease. Although 3-biased gene expression microarray platforms were the first generation to have widespread availability, newer platforms are gradually emerging that have more up-to-date content and/or higher cost efficiency. Deciphering the relative strengths and weaknesses of these various platforms for metabolic pathway level analyses can be daunting. We sought to determine the practical strengths and weaknesses of four leading commercially-available expression array platforms relative to biologic investigations, as well as assess the feasibility of cross-platform data integration for purposes of biochemical pathway analyses. METHODS: Liver RNA from B6.Alb/cre,Pdss2loxP/loxP mice having primary Coenzyme Q deficiency was extracted either at baseline or following treatment with an antioxidant/antihyperlipidemic agent, probucol. Target RNA samples were prepared and hybridized to Affymetrix 430 2.0, Affymetrix Gene 1.0 ST, Affymetrix Exon 1.0 ST, and Illumina Mouse WG-6 expression arrays. Probes on all platforms were re-mapped to coding sequences in the current version of the mouse genome. Data processing and statistical analysis were performed by R/Bioconductor functions, and pathway analyses were carried out by KEGG Atlas and GSEA. RESULTS: Expression measurements were generally consistent across platforms. However, intensive probe-level comparison suggested that differences in probe locations were a major source of inter-platform variance. In addition, genes expressed at low or intermediate levels had lower inter-platform reproducibility than highly expressed genes. All platforms showed similar patterns of differential expression between sample groups, with steroid biosynthesis consistently identified as the most down-regulated metabolic pathway by probucol treatment. CONCLUSIONS: This work offers a timely guide for metabolic disease investigators to enable informed end-user decisions regarding choice of expression microarray platform best-suited to specific research project goals. Successful cross-platform integration of biochemical pathway expression data is also demonstrated, especially for well-annotated and highly-expressed genes. However, integration of gene-level expression data is limited by individual platform probe design and the expression level of target genes. Cross-platform analyses of biochemical pathway data will require additional data processing and novel computational bioinformatics tools to address unique statistical challenges.
Cross-platform expression microarray performance in a mouse model of mitochondrial disease therapy.
Sex, Age, Specimen part, Treatment
View SamplesUnderstanding the transcriptional regulation of pluripotent cells is of fundamental interest and will greatly inform efforts aimed at directing differentiation of embryonic stem (ES) cells or reprogramming somatic cells. We first analyzed the transcriptional profiles of mouse ES cells and primordial germ cell (PGCs) and identified genes up-regulated in pluripotent cells both in vitro and in vivo. These genes are enriched for roles in transcription, chromatin remodeling, cell cycle and DNA repair. We developed a novel computational algorithm, CompMoby, which combines analyses of sequences both aligned and non-aligned between different genomes with a probabilistic segmentation model to systematically predict short DNA motifs that regulate gene expression. CompMoby was used to identify conserved over-represented motifs in genes up-regulated in pluripotent cells. We show that the motifs are preferentially active in undifferentiated mouse ES and Embryonic Germ cells in a sequence-specific manner, and that they can act as enhancers in the context of an endogenous promoter. Importantly, the activity of the motifs is conserved in human ES cells. We further show that the transcription factor NF-Y specifically binds to one of the motifs, is differentially expressed during ES cell differentiation and is required for ES cell proliferation. This study provides novel insights into the transcriptional regulatory networks of pluripotent cells. Our results suggest that this systematic approach can be broadly applied to understanding transcriptional networks in mammalian species.
Systematic identification of cis-regulatory sequences active in mouse and human embryonic stem cells.
Age, Specimen part, Time
View SamplesA new method for amplification and labeling of RNA is assessed that permits gene expression microarray analysis of formalin-fixed paraffin embedded tissue (i.e. FFPET) samples.
A novel method of amplification of FFPET-derived RNA enables accurate disease classification with microarrays.
Specimen part, Treatment
View SamplesWe show that aneuploidy is common in wild isolates of yeast, which are inherently tolerant to chromosome amplification and down-regulate expression at 40% of amplified genes. To dissect the mechanism of this dosage response, we generated isogenic strain panels in which diploid cells carried either two, three, or four copies of the affected chromosomes. Using a mixture of linear regression (MLR) model to classify genes, we find that expression is actively down regulated in proportion to increased gene copy at up to 30% of genes. Genes subject to dosage control are under higher expression constraint – but show elevated rates of gene amplification – in wild populations, suggesting that dosage compensation buffers copy number variation (CNV) at toxic genes Overall design: RNA-seq and transcriptome analysis of S. cerevisiae natural isolates having aneuploidy. Technical triplicate was performed for isogenic diploid strains having 2, 3 and 4 copies of a given chromosome (strain panels), while technical duplicate or singulate was performed on all other aneuploids.
Dosage compensation can buffer copy-number variation in wild yeast.
Subject
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