Project description:We performed ATAC-seq experiments for MDCKII 3D cells, confluent 2D cells as well as seeding day0 cells to study if transcriptome changes during epithelial morphogenesis is associated with chromatin changes.
Project description:Formation of epithelial tissues requires the generation of apical-basal polarity and the co-ordination of this polarity between neighboring cells to form a central lumen. MDCK cell line has proven to be a powerful model to study mammalian polarized epithelia in vitro. MDCK cells plated in extracellular matrix (ECM) form cysts, a spherical structure of polarized cells enclosing a central lumen which resembles epithelial tubular structures. The morphogenetic process requires drastic changes in cell architecture, which are regulated by change in gene expression. We used microarrays to identify genes up-regulated in lumen formation. The identification of up-regulated genes could lead us to characterize novel pathways needed for this process. MDCKII cells were plated in two different conditions: Cells cultured in confluence in plastic dishes, forming polarized monolayers (2D); or cells cultured in plastic dishes covered with Matrigel (ECM) forming three dimensional cysts (3D). Comparison of both transcriptomic profiles would lead us to identify up-regulated genes in the 3D condition, which would be good candidates to be key regulators of novel processes involved in lumen morphogenesis.
Project description:Comparative oncology is a developing research discipline that is being used to assist our understanding of human neoplastic diseases. Companion canines are a preferred animal oncology model due to spontaneous tumor development and similarity to human disease at the pathophysiological level. We use a paired RNA sequencing (RNA-Seq)/microarray analysis of a set of four normal canine lymph nodes and ten canine lymphoma fine needle aspirates to identify technical biases and variation between the technologies and convergence on biological disease pathways. Surrogate Variable Analysis (SVA) provides a formal multivariate analysis of the combined RNA-Seq/microarray data set. Applying SVA to the data allows us to decompose variation into contributions associated with transcript abundance, differences between the technology, and latent variation within each technology. A substantial and highly statistically significant component of the variation reflects transcript abundance, and RNA-Seq proved more sensitive for detection of transcripts expressed at low levels. Latent random variation among RNA-Seq samples is also distinct in character from that impacting microarray samples. In particular, we observed variation between RNA-Seq samples that reflects transcript GC content. Platform-independent variable decomposition without a priori knowledge of the sources of variation using SVA represents a generalizable method for accomplishing cross-platform data analysis. We identified genes differentially expressed between normal lymph nodes of disease free dogs and a subset of the diseased dogs diagnosed with B-cell lymphoma using each technology. There is statistically significant overlap between the RNA-Seq and microarray sets of differentially expressed genes. Analysis of overlapping genes in the context of biological systems suggests elevated expression and activity of PI3K signaling in B-cell lymphoma biopsies compared with normal biopsies, consistent with literature describing successful use of drugs targeting this pathway in lymphomas.
Project description:We performed time-course RNA-seq experiments for MDCKII 3D cell culture, and included seeding day0 cells and over-confluent 2D cells. To study how intracellular trafficking affects transcriptome changes, we also profiled trancriptome of DENND5A and AVL9 KD MDCKII cells by RNA-seq.
Project description:Comparative oncology is a developing research discipline that is being used to assist our understanding of human neoplastic diseases. Companion canines are a preferred animal oncology model due to spontaneous tumor development and similarity to human disease at the pathophysiological level. We use a paired RNA sequencing (RNA-Seq)/microarray analysis of a set of four normal canine lymph nodes and ten canine lymphoma fine needle aspirates to identify technical biases and variation between the technologies and convergence on biological disease pathways. Surrogate Variable Analysis (SVA) provides a formal multivariate analysis of the combined RNA-Seq/microarray data set. Applying SVA to the data allows us to decompose variation into contributions associated with transcript abundance, differences between the technology, and latent variation within each technology. A substantial and highly statistically significant component of the variation reflects transcript abundance, and RNA-Seq proved more sensitive for detection of transcripts expressed at low levels. Latent random variation among RNA-Seq samples is also distinct in character from that impacting microarray samples. In particular, we observed variation between RNA-Seq samples that reflects transcript GC content. Platform-independent variable decomposition without a priori knowledge of the sources of variation using SVA represents a generalizable method for accomplishing cross-platform data analysis. We identified genes differentially expressed between normal lymph nodes of disease free dogs and a subset of the diseased dogs diagnosed with B-cell lymphoma using each technology. There is statistically significant overlap between the RNA-Seq and microarray sets of differentially expressed genes. Analysis of overlapping genes in the context of biological systems suggests elevated expression and activity of PI3K signaling in B-cell lymphoma biopsies compared with normal biopsies, consistent with literature describing successful use of drugs targeting this pathway in lymphomas. RNA was extracted from 10 lymphoma fine needle aspirates attained from companion canines. 4 normal lymph node samples were obtained from a Beagle breeding colony at Pfizer, including two samples that were taken from the same dog but different lymph nodes. This Series represents the Affymetrix gene expression only, not RNA-Seq referenced above. RNA-Seq data have been submitted to SRA as SRA059558.