The impact of chronic temperature elevation on the spleen anti-viral transcriptomic response in Atlantic cod (Gadus morhua)
ABSTRACT: This experiment was designed to investigate the impacts of non-lethal increases in temperature on the anti-viral transcriptomic response of Atlantic cod. Selected genes identified as differentially expressed between samples injected with pIC but held at different temperatures were validated using QPCR. This was a common reference design. For the common reference each individual sample analyzed in the microarray experiment contributed with equal amounts of RNA to a pool. Sixty individuals were analyzed representing 6 biological replicates from 10 different experimental groups. Two separate control groups were used. Non-injected groups at the final temperatures (i.e. 10°C and 16°C) were used as prior to injection controls. At each time-point and temperatures there was also a sham-injected time matched control (injected with phosphate buffered saline [PBS]). The time points were 6 and 24 hours post-injection. Immunostimulated fish were injected with a weight-based dose of poly (I:C)(2 µg per g of wet body mass).
Project description:This study was performed to validate the newly developed CGP Atlantic cod 20K oligonucleotide microarray. Atlantic cod (Gadus morhua) received an intraperitoneal injection of either formalin-killed, atypical Aeromonas salmonicida (Asal) or PBS and transcriptional profiles of spleen tissues from Asal-injected fish were compared to those from pre-injection control fish and PBS-injected control fish. Gene expression profiles resulting from this study were compared to those from suppression subtractive hybridization library studies, that were previously performed on the same samples, and to literature. Gene expression patterns of single genes were confirmed by QPCR analysis. This study has shown that the newly developed CGP Atlantic cod 20K oligo microarray platform is a valuable tool for cod genomic research. Fish were assigned to 1 of 3 groups: Asal group, PBS group or 'undisturbed control' group (the latter were not handled during the experiment). These groups were kept in 3 separate tanks. For the test samples, RNA was used from individual spleen samples that were taken from 6 fish from each of 4 treatment groups: PBS group pre-injection (0H PBS), Asal group pre-injection (0H Asal), PBS group 24 hours post-injection (24HPI PBS) and Asal group 24 hours post-injection (24HPI Asal). All test samples were labeled with AlexaFluor 647. For the universal reference sample, RNA from 31 'undisturbed control' fish was pooled, with each individual contributing an equal amount, and labeled with AlexaFluor 555. Each individual test sample was hybridized together with the universal reference sample on an array. For one sample from the 0H PBS group the array failed. This study therefore includes 6 biological replicates for 0H Asal, 24HPI Asal and 24HPI PBS groups and 5 biological replicates for the 0H PBS group.
Project description:This experiment was designed to investigate the impacts of non-lethal increases in temperature on the anti-viral transcriptomic response of Atlantic cod. Selected genes identified as differentially expressed between samples injected with pIC but held at different temperatures were validated using QPCR. Overall design: This was a common reference design. For the common reference each individual sample analyzed in the microarray experiment contributed with equal amounts of RNA to a pool. Sixty individuals were analyzed representing 6 biological replicates from 10 different experimental groups. Two separate control groups were used. Non-injected groups at the final temperatures (i.e. 10°C and 16°C) were used as prior to injection controls. At each time-point and temperatures there was also a sham-injected time matched control (injected with phosphate buffered saline [PBS]). The time points were 6 and 24 hours post-injection. Immunostimulated fish were injected with a weight-based dose of poly (I:C)(2 µg per g of wet body mass).
Project description:Transcriptional profiling of infrarenal aortic tissue from Male 10-week-old C57BL/6J mice after AAA-induction with porcine pancreatic elastase, compared with sham-operated saline-injected mice. One day after AAA-induction, the mice were injected intraperitoneally with either lentiviral packaged miR-24 antagomir (anti-miR-24) or miR-24 mimic (pre-miR-24), or a scrambled microRNA control (scr-miR). Aortic samples were obtained 7 days after operation. The goal was to examine gene expression in developing AAA in this model, and to compare the effects of scr-miR, anti-miR-24 and pre-miR-24. Four condition experiment, one infrarenal aorta per array. Sham vs. scr-miR-PPE vs. anti-miR-24-PPE vs. pre-miR-24-PPE, all harvested at Day 7 post-operatively. After QC, the final analysis group (uploaded here) consisted of 18 arrays: Sham-Saline-treated (4 arrays), scr-miR-PPE-treated (5 arrays); pre-miR-24-PPE-treated (3 arrays); and anti-miR-24-PPE-treated (6 arrays).
Project description:Saccharomyces spp. are widely used for ethanol production however fermentation productivity is negatively affected by the impact of ethanol accumulation on yeast metabolic rate and viability. This study used microarray and statistical two-way ANOVA analysis to compare and evaluate gene expression profiles of two previously generated ethanol-tolerant mutants, CM1 and SM1, with their parent, S. cerevisiae W303-1A, in the presence and absence of ethanol stress. Although sharing the same parentage, the mutants were created differently; SM1 by adaptive evolution involving long-term exposure to ethanol stress, and CM1 using chemical mutagenesis followed by adaptive evolution-based screening. Compared to the parent, differences in the expression levels of genes associated with a number of GO categories in the mutants suggest that their improved ethanol stress response is a consequence of increased mitochondrial and NADH oxidation activities, stimulating glycolysis and energy production. This leads to increased activity of energy-demanding processes associated with the production of proteins and plasma membrane components, which are necessary for acclimation to ethanol stress. It is suggested that a key function of the ethanol stress response is restoration of the NAD+/NADH redox balance, which increases glyceraldehyde-3-phosphate dehydrogenase activity, and higher glycolytic flux in the ethanol-stressed cell. Both mutants achieved this by a constitutive increase in carbon flux in the glycerol pathway as a means of increasing NADH oxidation. Two conditions: 0% and 6.5 % (v/v) ethanol added to the culture growth medium for each strain. One set of triplicates with one dye swap for each condition. Each triplicate was prepared from different biological replicate (i.e. different culture).
Project description:Chytridiomycosis is an emerging infectious disease of amphibians caused by the chytrid Batrachochytrium dendrobatidis (Bd). The disease has been associated with global amphibian declines and is driving the species in the wild to extinction. Using DNA microarray technology we have analysed transcriptional changes in Xenopus tropicalis during the course (7 and 42 days) of infection by Bd under warm (26oC) and cold (18oC) temperatures.
Project description:Projected elevation of seawater temperatures poses a threat to the reproductive success of Caribbean reef-building corals that have planktonic development during the warmest months of the year. This study examined the transcriptomic changes that occurred during embryonic and larval development of the elkhorn coral, Acropora palmata, at a non-stressful temperature (28°C) and further assessed the effects of two elevated temperatures (30°C and 31.5°C) on these expression patterns. Using cDNA microarrays, we compared expression levels of 2,051 genes from early embryos and larvae at multiple developmental stages (including pre-blastula, blastula, gastrula, and planula stages) at each of the three temperatures. At 12 hours post-fertilization in 28°C treatments, genes involved in cell replication/cell division and transcription were up-regulated in A. palmata embryos, followed by a reduction in expression of these genes during later growth stages. From 24.5 to 131 hours post-fertilization at 28°C, A. palmata altered its transcriptome by up-regulating genes involved in protein synthesis and metabolism. Temperatures of 30°C and 31.5°C caused major changes to the A. palmata embryonic transcriptomes, particularly in the samples from 24.5 hpf post-fertilization, characterized by down-regulation of numerous genes involved in cell replication/cell division, metabolism, cytoskeleton, and transcription, while heat shock genes were up-regulated compared to 28°C treatments. These results suggest that increased temperature may cause a breakdown in proper gene expression during development in A. palmata by down-regulation of genes involved in essential cellular processes, which may lead to the abnormal development and reduced survivorship documented in other studies. Our experimental setup followed a reference design where all samples were hybridized against the same pool made up of equal amounts of RNA from all samples in the experiment. Biological duplicate samples were used for each temperature at each developmental time period. Common reference samples were labeled with Cy3 dye, while temperature samples were labeled with Cy5 dye. Microarrays for A. palmata contained 2,051 coding sequences, of which 54.3% had functional annotations as determined by homology to known genes.
Project description:Background Coral reefs are expected to be severely impacted by rising seawater temperatures associated with climate change. This study used cDNA microarrays to investigate transcriptional effects of thermal stress in embryos of the coral Montastraea faveolata. Embryos were exposed to 27.5C, 29.0C, and 31.5C directly after fertilization. Differences in gene expression were measured after 12 and 48 hours. Results Analysis of differentially expressed genes indicated that increased temperatures may lead to oxidative stress, apoptosis, and a structural reconfiguration of the cytoskeletal network. Metabolic processes were downregulated, and the action of histones and zinc finger-containing proteins may have played a role in the long-term regulation upon heat stress. Conclusions Embryos responded differently depending on exposure time and temperature level. Embryos showed expression of stress-related genes already at a temperature of 29.0C, but seemed to be able to counteract the initial response over time. By contrast, embryos at 31.5C displayed continuous expression of stress genes. The genes that played a role in the response to elevated temperatures consisted of both highly conserved and coral-specific genes. These genes might serve as a basis for research into coral-specific adaptations to stress responses and global climate change. The experimental setup followed a reference design, i.e. all samples were hybridized against the same pool made up of equal amounts of RNA from all samples. We used three technical replicates for each temperature. Common reference samples were labeled with Cy3, temperature samples with Cy5. Microarrays for M. faveolata contained 1,314 coding sequences, of which 43% had functional annotations as determined by homology to known genes.
Project description:Mesenchymal stem cells (MSC) omplexed to different transfection agents (R60, R200, DOSPER, jetPei, Pulsin). Two MSC populations (h21 and h24) and two passages (P2 and P4) were tested with different labelings. In a direct design experiment each sample was compared to its unlabeled control sample. Keywords: Human gene expression study Reference design with unlabeled cells in the Cy3 reference channel, and labeled cells in the Cy5 channel.
Project description:Circadian rhythms are daily oscillations of multiple biological processes driven by endogenous clocks. Imbalance of these rhythms has been associated with cancerogenesis in humans. To further elucidate the role of the circadian clock in cellular growth control, tumor suppression and cancer treatment, a standard of circadian gene regulations in healthy men is essential. Comparative microarray analyses were conducted investigating the relative mRNA expression of clock genes throughout a 24-hour period in biopsies obtained from oral mucosa of eight healthy diurnally active male study participants. A custom-designed oligo-based microarray which in addition to oncogenes and inflammatory genes contains probes for 20 clock genes and 70 clock-associated genes in human was used. Keywords: Human gene expression study Reference design with Cy3 labeled uniRNA and Cy5 labeled sample RNA.
Project description:Wound healing is associated with high rates of cell replication and lactate accumulation even under normoxic and hyperoxic conditions. Lactate accounts for various effects in tissue regeneration, such as collagen synthesis, angiogenesis, modulation of cytokine patterns and as recently shown for stem cell homing. Its influence on genes involved in cell replication has not been shown yet. Therefore, the effect of lactate considering genes involved in different cellular processes was investigated. Human umbilical vein endothelial cells (HUVEC) were cultured and incubated with lactate for different periods of time. Gene expression analysis was performed using custom-designed oligonucleotide microarrays. Keywords: Human gene expression study Reference design with Cy3 labeled uniRNA and Cy5 labeled sample RNA.