Microarray analysis of pig innate immune responses after experimental infection with Actinobacillus pleuropneumoniae
Ontology highlight
ABSTRACT:
ORGANISM(S): Sus scrofa
PROVIDER: GSE11404 | GEO | 2009/09/08
SECONDARY ACCESSION(S): PRJNA106531
REPOSITORIES: GEO
Similar Datasets
Project description:To study hepatic gene expression differences, liver samples from infected pigs (n = 10) were compared with liver samples from the non-infected control group (n = 5). Ten microarrays were performed, such that 5 randomly selected cDNA samples from the infected group were labeled with Oyster 550 (Genisphere Inc., Hatfield, PA, USA) and compared directly to 5 control animals labeled with Oyster 650 (Genisphere) analysing a pair of samples (one from an infected animal and one from a randomly chosen non-infected animal) on each microarray slide. The remaining five cDNA preparations from the infected group were labeled with Oyster 650 and compared in the same way to the five controls labeled with Oyster 550 on the remaining 5 microarray slides. This dye-swap design was applied to reduce variation due to dye effects and to provide as much biological replication as possible. Keywords: Disease state analysis Two-condition experiment, Disease vs. Control, Biological replicates: 10 disease replicates and 5 control replicates, 10 Microarrays comparing one disease replicate with one control replicate.
2009-09-08 | E-GEOD-11404 | biostudies-arrayexpress
Project description:The simultaneous measurement of gene expression for thousands of genes in a single analysis by the microarray technology allows researchers to describe transcriptomes in various samples of interest. Problems with variation in data quality derived from microarray experiments are well known and might result from poor RNA quality, background problems, or sub optimal signal strength. To assess variation due to the fluorescent dye chosen, three different dye pairs were tested for labelling of cDNA in gene expression analysis experiments on a porcine immune focused oligonucleotide microarray (POM3). This in-house oligonucleotide microarray allowed a direct comparison of background fluorescence, Median signal intensities, numbers of spots detected, and resistance to photobleaching between different dye pairs. We tested Alexa Flour 546/647, Cy3/Cy5 as well as Oyster 550/650 all from Genisphere Inc., Hatfield, PA, USA. Keywords: Comparison of fluorescent dyes Each dye pairs were hybridized on two slides. The same two samples were compared on all the slides used in the present study. The liver sample from a healthy animal was labeled with the high wavelength dyes (Oyster 650/Alexa 647/Cy5) and the liver sample from the sick animal was labeled with the low wavelength dyes (Oyster 550/Alexa 546/Cy3). Each slide was scanned 3 times, immediately after hybridization (first round), after 1 month of storage in darkness and after 24 hours on the bench (12 hours of daylight and 12 hours of artificial light). Total RNA from the infected and control liver samples were extracted using RNeasy midi kit (Qiagen, Denmark) and DNase treated using RNase-Free DNase Set (Qiagen). 3DNATM Array 900 expression array detection kits (Genisphere Inc.) were used for the labeling and cDNA synthesis reaction of the RNA in the present study. Three different pairs of fluorescent dyes Oyster 550/650 (Genisphere Inc.), Alexa 546/647 (Genisphere Inc.), and Cy3/Cy5 (Genisphere Inc.) were used in separate labeling reactions. Labeling was done according to the manufacturers protocol for large-scale cDNA synthesis. For the cDNA synthesis 9.2µg total RNA from each liver sample was used and mixed with 20U AmpliQ RTenzyme and 10x first strand buffer (Bie og Berntsen, Rødovre, Denmark). Hybridization and washing were performed according to the manufacturers instructions (Genisphere) using Corning hybridization chambers. For one cDNA hybridization reaction; 6.5µl cDNA from each synthesis, 0.5µl Salmon sperm (10µg/µl) and 13.5µl 2 x Formamide-Based Hybridization Buffer (3DNA Array 900, Genisphere) was used. A total hybridization mix of 29µl was applied under a 22I x 25 mm LifterSlip (Erie Scientific, Portsmouth, NH, USA) carefully avoiding air bubbles. Slides were incubated at 44oC in a water bath over night. Wash buffer 1 (3DNA Array 900, Genisphere) was preheated to 44oC for the post cDNA hybridization wash. For two 3DNA hybridization reaction mixes; 2.5µl of each Capture reagent and 26µl SDS-Based hybridization buffer was mixed to a final volume of 52µl. 26µl 3DNA hybridization mix was applied to each slide, and incubated in darkness in a water bath at 50oC for 4 hours. Wash buffer 1 was preheated to 60oC for the post 3DNA hybridization wash. Slides were scanned on a CCD ArrayWoRxe auto (Applied Precision, Issaquah, WA, USA) using different exposure times (0.3 for 595 nm and 1.2 for 685 nm).
2010-05-06 | E-GEOD-4855 | biostudies-arrayexpress
Project description:The simultaneous measurement of gene expression for thousands of genes in a single analysis by the microarray technology allows researchers to describe transcriptomes in various samples of interest. Problems with variation in data quality derived from microarray experiments are well known and might result from poor RNA quality, background problems, or sub optimal signal strength. To assess variation due to the fluorescent dye chosen, three different dye pairs were tested for labelling of cDNA in gene expression analysis experiments on a porcine immune focused oligonucleotide microarray (POM3). This in-house oligonucleotide microarray allowed a direct comparison of background fluorescence, Median signal intensities, numbers of spots detected, and resistance to photobleaching between different dye pairs. We tested Alexa Flour 546/647, Cy3/Cy5 as well as Oyster 550/650 all from Genisphere Inc., Hatfield, PA, USA. Keywords: Comparison of fluorescent dyes
2009-01-16 | GSE4855 | GEO
Project description:Chickens were kept under a regimen of 12 hours of light and 12 hours of dark for 7 days. At LD2, 2 hours into the light cycle on the 8th day, 3 chickens were sacrificed by decapitation and their retinas dissected and pooled. Similarly, at LD18, 6 hours into the dark cycle on the 8th day, retinas were harvested from 3 chickens. Total RNA was extracted from each set of retinas using Trizol Reagent (Invitrogen). The total RNA samples were amplified to produce aRNA using the Message Amp Kit (Ambion). Randomly primed fluorescent probes were produced from the aRNA samples using a Genisphere 3DNA Array 900MPX expression array detection kit. The fluorescent dye on the probe derived from the experimental aRNA (LD2) was Cy5 while the dye on the control probe (LD18) was Cy3. Hybridizations and washes were as suggested by Genisphere. Labeled arrays were scanned in an Affymetrix 428 array scanner. The images obtained were subsequently analyzed by GenePixPro software (Axon Instruments). The GenePix results (.gpr) file was further analyzed by GeneSpring 5 (Silicon Genetics) which applied intensity dependent LOWESS normalization to the results and calculated normalized Cy5 to Cy3 ratios.
2010-12-10 | GSE25992 | GEO
Project description:Necrotizing enterocolitis (NEC), a serious gastrointestinal disease that afflicts 5-10% of preterm infants, often progresses rapidly from mild food intolerance into extensive haemorrhage, inflammation and necrosis. Events leading to NEC have remained poorly defined. Similar disease characteristics are observed in preterm pigs 24-48 h after feeding formula. Using this model, we aimed to characterize the temporal development of NEC, and describe the functional and immunological response of the preterm intestine preceding NEC. Keywords: time course Pigs from treatment groups TPN (n=5), and 8 h (n=5) and 24 h (n=5-6) FORM and COLOS were randomly selected for microarray analysis. Equal amounts of total distal small intestinal RNA from all pigs was pooled to make the reference sample. Samples and reference pool were labelled with Oyster 550 and 650, respectively. The in-house spotted porcine oligonucleotide microarray version 4 (POM4) is a low density microarray consisting of 384 different oligonucleotide probes representing more than 200 different immune related genes and eight different array control oligonucleotides (ArrayControl; Ambion, Nærum, Denmark). The immunologically relevant 60-70mer oligonucleotide probes represent interferons and interleukins (and receptors), chemokines (and receptors), acute phase proteins, apoptosis-related factors and sequences with relevance to Toll-like receptors and their intracellular signalling pathways.
2010-05-07 | E-GEOD-13516 | biostudies-arrayexpress
Project description:Preterm neonates are susceptible to gastrointestinal (GI) disorders such as necrotizing enterocolitis (NEC). Maternal milk, and especially colostrum, protects against NEC via growth promoting, immunomodulatory and antimicrobial factors. The fetal enteral diet, amniotic fluid (AF), contains similar bioactive components and we hypothesized that postnatal AF administration would reduce inflammatory responses and NEC in preterm neonates. Thirty preterm pigs (92% gestation) were delivered by caesarean section and fed total parental nutrition (TPN) for 48 h followed by enteral porcine colostrum (COLOS, n=7), infant formula (FORM, n=13) or formula + porcine AF (AF, n=10). Using a previously validated model of NEC in preterm pigs, we determined the structural, functional, microbiological and immunological responses to AF when administered prior to and after introduction of a suboptimal enteral formula diet. Keywords: Healthy versus inflammed tissues in relation to necrotizing enterocolitis Pigs from each treatment group (COLOS, n=4; FORM, n=6; and AF, n=7) were randomly selected for microarray analysis of frozen distal small intestine samples. The FORM group was further divided into formula-fed healthy pigs (F-HEA, n=3) and formula-fed NEC pigs (F-NEC, n=3) in order to compare sick versus healthy formula fed pigs. Equal amounts of total distal small intestinal RNA from all pigs were pooled to make the reference sample. Samples and reference pool were labelled with Oyster 550 and 650, respectively. The in-house spotted porcine oligonucleotide microarray version 4 (POM4) is a low density microarray consisting of 384 different oligonucleotide probes representing more than 200 different immune related genes.
2010-05-06 | E-GEOD-13515 | biostudies-arrayexpress
Project description:Gene expression profiling of human promyelocytic cells in response to infection with Anaplasma phagocytophilum. Total RNA derived from 3DPI Anaplasma phagocytophilum-infected HL-60 cells was labeled with A647 and total RNA derived from 3DPI Mock-infected HL-60 cells was labeled with A546. For each, 5 µg of total RNA was labeled using Genisphere Array900, Alexa Fluor dyes and SuperscriptII. Slide scanned with ScanArray Express and images processed with GenePix Pro version 4.0. Normalized log ratios VALUES determined using R-project statistical environment (http://www.r-project.org) and Bioconductor (http://www.bioconductor.org) through the GenePix AutoProcessor (GPAP, http://darwin.biochem.okstate.edu/gpap) website (P. Ayoubi, unpublished results). Keywords: time-course
2005-02-25 | GSE1694 | GEO
Project description:The goal of this study is to gain a better understanding of the transcriptional responses of M. hyopneumoniae under norepinephrine stress. Six independent RNA samples from norepinephrine-exposed cultures were paired with six independent RNA samples from control cultures for hybridization to six two-color microarrays. For three arrays, the control RNA sample was labeled with Cy3 dye and the experimental RNA sample was labeled with Cy5 dye, the dyes were reversed for the other three arrays to overcome dye bias. Keywords: stress response
2008-03-01 | GSE8494 | GEO
Project description:Chickens were kept under a regimen of 12 hours of light and 12 hours of dark for 7 days. At LD2, 2 hours into the light cycle on the 8th day, 3 chickens were sacrificed by decapitation and their retinas dissected and pooled. Similarly, at LD18, 6 hours into the dark cycle on the 8th day, retinas were harvested from 3 chickens. Total RNA was extracted from each set of retinas using Trizol Reagent (Invitrogen). The total RNA samples were amplified to produce aRNA using the Message Amp Kit (Ambion). Randomly primed fluorescent probes were produced from the aRNA samples using a Genisphere 3DNA Array 900MPX expression array detection kit. The fluorescent dye on the probe derived from the experimental aRNA (LD2) was Cy5 while the dye on the control probe (LD18) was Cy3. Hybridizations and washes were as suggested by Genisphere. Labeled arrays were scanned in an Affymetrix 428 array scanner. The images obtained were subsequently analyzed by GenePixPro software (Axon Instruments). The GenePix results (.gpr) file was further analyzed by GeneSpring 5 (Silicon Genetics) which applied intensity dependent LOWESS normalization to the results and calculated normalized Cy5 to Cy3 ratios. 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.
2010-12-10 | E-GEOD-25992 | biostudies-arrayexpress
Project description:The purpose of the McrBC methylation microarray assay is to determine which regions of a genome are methylated versus those that are unmethylated in a single Arabidopsis thanliana genotype. McrBC is a methylation-sensitive enzyme that restricts DNA only at purine-Cmethyl half sites when separated between 50bp and 3kb. A designated amount of DNA from a particular genotype is sheared to a size range of 1kb-10kb using nebulization. We restrict half of the nebulized DNA with McrBC, and the methylated fraction is then removed from the unmethylated fraction through gel purification of DNA fragments greater than 1kb.* The remaining nebulized DNA is subjected to the same gel purification scheme, but with no McrBC treatment. In a single hybridization, the untreated sample is labeled with Cy5 and the McrBC-treated sample with Cy3. Thus, after labeling and microarray hybridization, the ratio of normalized Cy5 to normalized Cy3 represents the relative methylation at the sequence represented by the spot on the microarray. Dye swap analysis is carried out to take account of experimental variation by repeating the hybridization with identical samples labeled with Cy3 and Cy5, respectively. The two samples in this series are complementary hybridizations in a dye-swap analysis. This is the normalized result of the paired dye swap samples VC121 and VC127. The ANOVA model of Kerr, Martin and Churchill (2000) was used to analyze the data from the dye-swap experiments, with terms included to account for gene, dye-by-gene, treatment-by-gene, and random error terms. These samples were not subjected to hypothesis testing. Therefore, P-values are reported as -1 and no features are flagged as significantly methylated. Keywords: other
2004-06-12 | GSE1329 | GEO