Project description:Human hepatocellular carcinoma cells, HepG2 , cultured in DMEM containing 10% fetal calf serum. To study gene expression changes induced by Vitamin K2, total RNA was isolated following the Isogen procedure (Nippon-gene, Tokyo, Japan) from HepG2 cells with and without 50 microM of Vitamin K2 treatment for three days. The cDNA microarrays Human Oligo Chip 30K “AceGene” subset A was purchased from Hitachi Software Engineering Co. (Yokohama-City, Japan). This array contains 10368 kinds of gene-specific 50 mer sense oligonucleotides. Subset A contains mainly known function ORF oligo probes. Preparation of fluorescent cDNA targets by an indirect labeling approach was performed using PowerScript reverse transcription kit (Clontech). First-strand cDNAs were synthesized from 30 microg of total RNA from HepG2 cells treated with or without Vitamin K2 using random primer and PoweScript Fluorescent Labelling Kit. Monofunctional, N-hydroxysuccinimide-activated fluorescent dyes (Cy3 and Cy5; Amersham) were coupled to the cDNAs by reaction with the amino functional groups. Untreated cells were labeled with Cy3 as a control and treated cells with Cy5 as a sample. After free dyes were removed using MinElute Purification kit (Qiagen), the targets were mixed together and added to the microarrays, and then incubated overnight (16 hours) at 42?C. The slides were washed at 30?C in each with 2x SSC, 0.1% SDS and 2x SSC for 10 min and 5 min, respectively, and washed in 1x SSC for 5 min. Fluorescent images of the hybridized microarrays were scanned with a fluorescence laser confocal slide scanner (Affymetrix 428 Array Scanner, Affymetrix, Santa Clara, CA). The Cy3 and Cy5 intensities with background subtraction were determined by ImaGene 4.2 software (BioDiscovery, Marina Del Rey, CA). The expression data were then filtered based on their channel intensities, spot size and flag (missing or inaccurate data), and the Cy5/Cy3 ratios were calculated and normalized by median-centering the log-ratio of all genes. This GEO Series was created by the GEO staff as part of a cleanup effort to ensure that all GEO Samples are included within a Series entry.

Project description:The transcriptional response of Atlantic salmon (Salmo salar) to infectious hematopoietic necrosis (IHN) virus was elucidated using 16,008 gene GRASP cDNA microarrays. S. salar were exposed to the IHN virus in a waterborne challenge, and kidney samples from five fish sampled on each of days 0, 1, 5 and 9 were analysed in the microarray study. Validation of nine of the significant genes was conducted on fish sampled on alternate days from 1 to 13 and compared to the unchallenged control sampled on day 0. The key pathways up-regulated in response to the virus included endosomal transport, type I and II interferon responses, the alternative complement pathway, apoptosis, phagocytosis and phagocytic cell oxidation, natural killer cell activity, antiviral replication via iron sequestering, virally-induced disruption of the cell cycle, retroviral DNA integration, T-cell activation and cellular immunity. Fish that did not contain viral titer on days 5 and 9 responded in a similar manner to those with titer, but further induced a number of genes involved in anti-viral replication (Glutathione peroxidase 1 and ferritin H), anti-proliferation (PTEN and B-cell receptor associated protein 32), lysosomal response (CD63 antigen), pro-inflammatory response (lipopolysaccharide binding protein, stress activated JNK1, thiol peroxidase), and T-cell activation (T-cell activation Rho-GTPase activating protein) that may increase resistance to the virus. Three genes that were potentially co-opted by the virus to enhance infectivity were also identified, including uPAR (angiogenesis), CypA (viral replication and infectivity), and BAF1 (viral protein biosynthesis). Perhaps the most notable finding was the up-regulation of uPAR which can function to increase the density of fibronectin, the receptor of the IHN virus, on the cell surface, hence facilitate viral entry into the cell. Keywords: Immunlogical response to IHN virus challenge A preliminary microarray experiment was run on the waterborne-challenged S. salar whereby the five individuals sampled on a given day were combined and run on a single slide, and we compared the expression profiles from each day (days 0 (unchallenged control) through 11). From this experiment, we noted that the most profound changes were occurring around days 1, 5, and 9, so we performed subsequent experiments at these time points, with five fish per day and 5 control fish analysed in total.

Project description:A large-scale functional genomics study revealed shifting energy generating processes in white muscle during the final 1,300 km migration of wild sockeye salmon to their spawning grounds in the Fraser River, British Columbia. In 2006, Lower Adams stock sockeye salmon ceased feeding after passing the Queen Charlotte Islands, 850 km from the Fraser River. Enhanced protein turnover and reduced transcription of actin, muscle contractile and heme-related proteins were early starvation responses in saltwater. Arrival to the estuarine environment triggered massive protein turnover through induction of proteasoma and lysosomal proteolysis and protein biosynthesis, and a shift from anaerobic glycolysis to oxidative phosphorylation. Response to entry into freshwater was modest, with up-regulation of heat shock proteins and nitric oxide biosynthesis. High river temperatures resulted in a strong defense/immune response and high mortalities in 50% of fish. Arrival to the spawning grounds triggered further up-regulation of oxidative phosphorylation and proteolysis, down-regulation of protein biosynthesis and helicase activity, and continued down-regulation of muscle proteins and most glycolytic enzymes. However, sharp up-regulation of PFK-I indicated induction of glycolytic potential at the spawning grounds. The identification of potential environmental cues triggering genome-wide transcriptional shifts in white muscle associated with migration and the strong activation of proteasomal proteolysis were both novel findings. Keywords: Functional genomics study on wild-caught fish The experiment was based on expression profiles of white muscle tissue collected from wild migrating adult sockeye salmon during their return spawning migration back to the Fraser River. Fish were collected from seven sites along the final 1,300 km migration path, and white muscle samples were quickly frozen in liquid nitrogen upon capture. Marine sampling sites included (from north to south) the Queen Charlotte Islands (QCI), Johnstone Strait (JS), Juan de Fuca Strait (JDFS), and the Strait of Georgia (SOG). Freshwater sampling sites included Whonnock (W), Savona (SV) and the Lower Adams Spawning Grounds. Genetically-based stock ID was used to identify the natal sites of fish collected from the wild. The experiment was designed to profile the transcriptional shifts associated with migration of the Adams River stock complex. The total experiment included 80 microarray slides, with a minimum biological replicate size per site of 6 (SV), and maximum of 18 (JS) (see supplemental table for details). Additional intra-site variables, which could only be addressed in some sites, included sex (female biased) and river entry timing (for JS, JDFS and W sites; identified through radio-tracking of marine collected fish). Total RNA was amplified (1 round) with MessageAmpTMII-96 kit (Ambion, TX, USA), and reverse transcribed to cDNA before labelling with ALEXA dyes using the Invitrogen Indirect Labelling Kit. The experiment was based on a reference design, with the reference containing the combined aRNA of all individuals used in the experiment. Individual samples were labelled with Alexa 555 and the reference control with Alexa 647, with no dye flips included. A single technical replicate of one SV fish (replicate 5) was included in the experimental design. This experiment is part of a larger white muscle experiment containing additional sockeye salmon stocks.

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