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: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:Acute myeloid leukemia (AML) is the most common and severe acute leukemia in adults. It is a heterogeneous disease where the subset of molecularly different types, presenting various morphological features and differentiation stage, can be distinguished. Genomic research of leukemias is conducted since 1999 and large cohort studies shown that particular genetic alterations correspond with specific gene expression signatures. However, not always they provide clinically relevant information. The most unknown group is cytogenetically normal acute myeloid leukemia (CN-AML, 40-49% of all AML cases). The aim of our experiment was to determine selected gene expression profiles in CN-AML, using small, boutique microarray. The array contained 933 oligonucleotide probes, mainly complementary to acute myeloid leukemia markers, genes involved in leukemic transformation and myeloid cell proliferation, differentiation and maturation. Our test dataset included 40 hybridizations: 24 corresponding with blood and bone marrow samples collected from 12 patients with AML M1 or M2 FAB subtype and 16 corresponding with healthy control samples. Total RNA was extracted from the mononuclear cell fractions, reversibly transcribed to cDNA and labeled with Alexa 647 dye. The common reference was RNA isolated from HL-60 cell culture, labeled with Alexa 555 dye.
Project description:Drought-treated and corresponding control root tissue of poplar was subjected to array analyses Two-condition experiment, control (K) vs. Drought-stressed (S) leaves. Biological replicates: 3 control (1-3), drought-exposed (1-3), independently grown and harvested. One swap replicate per array.
Project description:Transcriptional profiling of larval stage I - IV of Homarus americanus, for assessment of stage specific developmental gene expression. Individuals from each stage were gathered in July of 2009 and 2010. A reference design was used to permit comparison of all stages. Study aims included assessment of global gene expression of larval development of healthy larvae, and identifying novel molecular pathways involved in H. americanus development. Four condition experiment, stage I - IV individuals: 10 biological replicates per stage. Stages collected independantly from each other; sampling conducted over 2 seasons. Reference sample prepared from pooled RNA of 4 individuals per stage.
Project description:A total of 163 genes were found to be differentially expressed; 38 genes of them were up regulated and 125 genes were down regulated. Most notably the functional categories that were affected include Virulence genes (18 genes 11% of the total significantly differentiated genes), carbohydrate metabolism and cell envelop realted genes ( 31, 19.1% of the total), and aminoacid transport genes (21genes, 12.9%). Among the virulence-related genes, the most notable one were those belong to the production of capsule, the M protein, exotoxin, NAD glycohydrolase and Streptolysin S. Some of the capsule related genes were down regulated by more than 64 fold. These results indicate that CdhA (group A Streptococcal Cell division controlling and Chain-forming cell wall hydrolase) plays an important role in the regulation of virulence. S.pyogenes strain SF370 (wild-type) was procured from ATCC (ATCC 700294). M1-CdhA(-) mutant strain was obtained using pFW5 vector containing spectinomycin resistance marker (aad9). The mutant lacking CdhA is highly defective in cell division and growth, lacks antiphagocytic property and attenuated for virulence. Both strains (M1-Wild-type and M1-CdhA(-) were grown in Todd-Hewitt broth to their late log phase. Bacteria were harvested by centrifugation and washed twice with sterile PBS. Total RNA was isolated from these washed streptococci using Qiagen RNeasy Mini kit. For synthesis and labeling of cDNA, 20 ug of total RNA, 1.5 ug of random hexamer, 0.5mM dNTPs (except that 0.2mM of dTTP was replaced by the same amount of amino-allyl dUTP to incorporate dUTP into first-strand cDNA) were used in each reverse transcriptase reaction (Superscript II Reverse Transcriptase, Invitrogen). Purified cDNA preparations from the wild-type and M1CdhA(-) mutant strains were labeled with either Alexafluor-555 or Alexafluor-647 depending on the experimental design ( i.e. dye swap experiment). Differentially labeled probes were then combined and purified. Using three independently isolated RNA preparations (biological replicates), a total of 11 experiments (incorporating dye swaps) were performed. Accordingly eleven hybridization measurements for this mutant were performed. Thus Exp-1 to -4 (GSM388703, GSM388717, GSM388718 and GSM388719) are the technical replicates of the biological sample-1, Exp-5 to 8(GSM388732, GSM388733, GSM388734, and GSM388735) are technical replicates of biological sample-2 and Exp-9,-10, and -11 (GSM388736,GSM388737, and GSM388738) are technical replicates of the biological sample-3. Signals of the bound reagents on the microarray spots in terms of relative fluorescence values were measured and quantified by a laser scanner (GenePix 4100 ) at 10um/pixel resolution. The resulting images were processed using Gene Pix Pro software (version 4.0, Axon instruments). The raw data were obtained in the form of GenPix *.gpr output files. The web application CARMAweb (Comprehensive R based microarray analysis web service) was used for the normalization and analysis of microarray data. All raw data (in the form of *. GPR files) were uploaded to the web application in the data directory. Using appropriate navigation tree, background correction from the foreground signal was applied and within microarray normalization was achieved using the Lowess method. Genes flagged as bad spots by the scanning software were excluded from the analysis and all flagged spots were given a weight of zero. The normalized data were then subjected to fold-change analysis and t-statistics using Bioconductor multtest package. CARMAweb allows to set Log2 cut-off values for both the M (regulation) and the A (average expression) values. Differentially expressed genes (Log2 values of Mutant647-red vs. wild-type555-green or Log2 values Mutant-555(green) vs Wild-type 647-red) were defined based on cut-off value >1 Log2< i.e. all genes that show a two or more-fold up- or down-regulation. Bioconductors Multtest package provided suitable method to adjust P values according to multiple hypothesis testing problems.
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).