Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Ciprofibrate stimulated gene expression in rats

ABSTRACT: Samples and RNA preparation: The series is composed of 4 hybridizations for analysis of differentially expressed genes in the liver of ciprofibrate dosed vs. control rats. Rats (200-250 g) were given (by gastric intubation) tap water (control group, n=5) or ciprofibrate (50 mg/kg body weight, once daily for 60 days, n=4). Liver total RNA extraction was performed first by ultracentrifugation on a cesium chloride cushion and then using TRIZOL Reagent (GIBCO BRL Life Technologies, New York, NY). Labeling and hybridization: One microgram of total RNA from the control pool (n=5), and from each ciprofibrate dosed rat was reverse transcribed and labeled with Cy3- and Cy5-attached dendrimer, respectively, using 3DNA Array Detection Submicro kit as described in the manufacturer's protocols (Genisphere, Montvale, NJ). The labeling reactions were also spiked with exogenous control RNAs (Stratagene) at expected Cy5/Cy3 ratios of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 0.5, 0.33, 0.25 and 0.125 for control spike 1, 2, 3, 4, 5, 6, 8, 9, 10 and 7, respectively. The Cy3 and Cy5 labeled samples (combined volume 5 microliter) were mixed with 30 microliter of the hybridization solution containing 0.25 M NaPO4, 1 mM EDTA, 4.5% SDS, 1 x SSC, 2 x Denhardt's solution, 0.25 microliter antifade reagent (Genisphere) and 0.2 microgram mouse COT-1 DNA (GIBCO BRL Life Technologies), added onto the array and covered with 22 x 50mm cover slip. The array was assembled into humidified hybridization chamber (Corning) and hybridized at 65oC for 18 h by submerging in a water bath. Post-hybridization washes were done once in 2 x SSC, 0.2% SDS at 55oC for 15 min, then in 2 x SSC at room temperature for 15 min, and finally with 0.2 x SSC at room temperature for 15 min. Scanning, image analysis and data processing: Arrays were scanned at 10 micrometer resolution with a confocal laser scanner constructed in-house in collaboration with NEMKO (Trondheim, Norway) according to a prototype developed at NHGRI (http://www.nhgri.gov/DIR/LCG/15K(HTML/). Microarray image analysis was done with MicroArray Suite software version 2.1 with globally normalization (Scanalytics, Inc., Fairfax VA). First, spots undetected (target intensity=1) in at least one array were excluded. Then, spots with signal intensity (averages of the 4 arrays) less than 300 in both channels were removed. The fluorescence intensity ratios (Cy5/Cy3) for genes measured from probes spotted in duplicate were averaged for each slide. Then, mean ratios of the 4 hybridizations were calculated for each gene, and differentially regulated genes were determined using 1.4 fold change cutoff values (1.4 for up-regulated or 0.7 for down-regulated genes).

ORGANISM(S): Rattus norvegicus

SUBMITTER: I Bakke

PROVIDER: E-GEOD-335 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Liver gene expression in rats in response to the peroxisome proliferator-activated receptor-alpha agonist ciprofibrate.

Yadetie Fekadu F Laegreid Astrid A Bakke Ingunn I Kusnierczyk Waclaw W Komorowski Jan J Waldum Helge L HL Sandvik Arne K AK

Physiological genomics 20030929 1

Fibrate class hypolipidemic drugs such as ciprofibrate activate the peroxisome proliferator-activated receptor-alpha (PPARalpha), which is involved in processes including lipid metabolism and hepatocyte proliferation in rodents. We examined the effects of ciprofibrate (50 mg/kg body wt per day for 60 days) on liver gene expression in rats using cDNA microarrays. The 60-day dosing period was chosen to elucidate both the metabolic and proliferative actions of this substance, while avoiding confoun ...[more]

PMID: 12851464

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Project description:Although immune mechanisms have been described for Peyer’s patch function, in discriminating food nutrients and commensal microflora from pathogenic challenge, an understanding of gene expression patterns associated with normal intestinal homeostasis is lacking. The effects of commensal bacterial colonization on global gene expression in the small intestine of germ-free mice have been described (1, 2). However, no studies have compared expression patterns of various healthy lymphoid tissues in conventionally-raised pigs or other species. The objective of this study was to investigate the pattern of normal gene expression in jejunal Peyer’s patches (JPP) of healthy pigs. We hypothesized that gene expression of intact jejunal Peyer's patch is characterized by genes associated with absorption and secretory functions as well as genes associated with GALT-specific immune functions. Eight hybridizations were performed, with dye swaps of JPP mRNA from four juvenile pigs. Each hybridization compared JPP from an individual pig to the jejunal mesenteric lymph node reference sample, which was pooled from all juvenile pigs in the study. However, two of the hybridizations resulted in poor quality data, and were excluded from further analysis. Normalization, hierarchical clustering, data visualization, and statistical analysis were performed by GeneSpring version 6.2 (Silicon Genetics, Redwood, CA). GeneSpring standardized the intensities for each spot by subtracting the local background, and then normalized globally by locally weighted linear regression (Lowess). A Lowess curve was fit to the log-intensity versus log-ratio plot. 20.0% of the data was used to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. If the control channel was lower than 10 then 10 was used instead. Spots with a flag value of “0” were excluded. The intensity of negative control DNA-containing spots was used to additionally exclude low intensity data. Negative controls include the gene for Arabidopsis thaliana homeodomain-like protein (AY054571); 200 ng/microl Poly(dA) (Amersham, Piscataway, NJ); a 461 bp fragment of the kanamycin-resistant gene, amplified from the pET24b+ plasmid (Novagen, Madison, WI); PRRS virus strain VR-2332 (PRU87392) ORFs 2, 3, 4, 5, 6 and 7; pCMVSport 6 plasmid (Invitrogen, Carlsbad, CA); and pGEM-T plasmid (Promega, Madison WI). The mean intensity of the negative controls for both Cy3 and Cy5 was calculated for each slide, and spots with a mean intensity less than the mean intensity + 2SD were excluded from further analysis. After normalization, GeneSpring averaged replicate spots for each clone across hybridizations. GeneSpring used the Cross Gene Error Model, which was based on replicate values. A Student’s t-test with the Benjamini and Hochberg false discovery rate multiple testing correction (MTC) verified the difference between the natural log of the normalized gene expression ratio (JPP:j-MLN) and a ratio of 1.0. p-values less than 0.05 were considered significant. Series_references: 1.Fukushima K, Ogawa H, Takahashi K, Naito H, Funayama Y, Kitayama T, Yonezawa H, and Sasaki I. Non-pathogenic bacteria modulate colonic epithelial gene expression in germ-free mice. Scand J Gastroenterol 38: 626-634, 2003. 2. Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, and Gordon JI. Molecular analysis of commensal host-microbial relationships in the intestine. Science 291: 881-884, 2001.

Project description:Batch cultures of Wild-type C. jejuni NCTC11168 were grown in 150 ml volumes of Mueller-Hinton broth in 250 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10 % O2, 10 % CO2, 80 % N2) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 42 ÂºC. When mid-exponential phase was reached 0.25 mM GSNO was added to half of the cultures. After a 10 minute exposure samples of both treated and untreated cells were harvested into phenol/ethenol to stabilize the RNA and total RNA was purified using Qiagenâs RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis. A C. jejuni cgb mutant was grown in two parallel custom-built chemostats based on Sigma Proculture Dynalift spinner flasks with a working volume of 125 ml, and the dilution rate (which at steady state is equal to the specific growth rate) of 0.1 h-1. A gas mix of 10% O2, 10% CO2 and 80% N2 was passed into the head space of the culture vessel at a rate of 0.25 l min-1 to obtain a microaerobic atmosphere, and silicone water jackets maintained the temperature at 42 ÂºC. The flasks were placed on KMO 2 basic IKA-Werke stirrers (arbitrary setting, 260; Scientific Laboratory Supplies), which gaveeffective transfer of O2 from the gaseous atmosphere to the culture by virtue of a stable vortex. A fresh overflow sample was used to check the pH at the time of harvest (which was consistently between 6.6 and 7) and to ensure that there was no contamination by plating a 20 Î¼l sample onto both nutrient agar plates incubated at 37 Â°C at atmospheric oxygen, and Mueller-Hinton plates incubated at 42 Â°C in the microaerophilic work station. When staedy-state was reached, as verified by collecting at least 5 culture volumes, GSNO (final concentration 0.25 mM) was added to one culture. After a further 10 min the 125 ml volume was separated into 3 equal volumes and each was mixed immediately on ice with 3.56 ml 100% ethanol and 185 ml phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier. cDNA synthesis was carried out using 12 Âµg of starting material, which was primed with 9 Âµg of pd(N)6 random hexamers (Amersham Biosciences). Reaction mixtures (20 Âµl) containing 0.5 mM ATP, 0.5 mM TTP, 0.5 mM GTP, 0.2 mM CTP, and 1 mM Cy3- or 1 mM Cy5-dCTP were incubated for 2 h at 42 Â°C with 200 U of Superscript II RNase H reverse transcriptase (Invitrogen). Following synthesis, cDNA was purified by using a PCR purification kit (Qiagen) to remove the unincorporated deoxynucloside triphosphates, fluorescent dye, and primers. Equal volumes of cDNA were combined and evaporated to near dryness with an SPD121P Speed Vac (Thermon Savant). For hybridisation to the microarray slides, cDNA was resuspended in an appropriate volume of salt-based hybridization buffer (provided by Ocimum Biosolutions). Prior to addition to the slides, cDNA samples were heated to 95 Â°C for 3 min, and then placed on ice for no more than 3 min. The slides were placed in MWG Biotech hybridization chambers and incubated for 16 to 24 h in a shaking water bath at 110 rpm and 42 Â°C. Following incubation , the slides were washed sequentially for 5 min in 2x SSC-1 % sodium dodecyl sulphate, 1x SSC, 0.5x SSC and 0.1x SSC at 37 Â°C with gentle agitation (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The slides were dried by centrifugation at 254 xg for 5 min and subsequently scanned with an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained by using commercially available software from Biodiscovery, Inc. (Imagine version 4.0, and Genesight, version 3.5). The mean values from each channel were log2 transformed and normalized by using the subtract-by-mean method to remove intensity-dependent effects in the log2 (ratio) values. The Cy3/Cy5 fluorescence ratios were calculated from the normalized values. Biological experiments (i.e. chemostat growth with and without 0.25 mM GSNO addition) were carried out three times and a dye swap was performed for two of the three experiments which provided two technical repeats, one each for two of the three biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed â¥ twofold and displayed and P value of â¤ 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.

Project description:Wild-type Campylobacter jejuni NCTC11168 was grown in 2 homemade, parallel chemostats. Cells were grown in 125 ml volumes of Mueller-Hinton Broth where the growth rate was controlled by the dilution rate (0.1 h-1). A gas mix of 80% nitrogen, 10% carbon dioxide and 10% oxygen was pumped into the head-space of the vessels at a rate of 0.25 l/min to obtain a microaerobic environment and the culture was maintained at 42 ÂºC via a water jacket. Cells were grown as above until steady-state had been reached. At steady-state one of the cultures was exposed to 10 uM NOC-5 & -7 (final concentration) for a period of 10 min. Samples of both the control and stressed cultures were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using Qiagenâ??s RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis. Equal quantities of RNA from control and GSNO-supplemented cultures were labelled using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 4). The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. C. jejuni NCTC11168 was grown in two parallel custom-built chemostats based on Sigma Proculture Dynalift spinner flasks with a working volume of 125 ml, and the dilution rate (which at steady state is equal to the specific growth rate) of 0.1 h-1. A gas mix of 10% O2, 10% CO2 and 80% N2 was passed into the head space of the culture vessel at a rate of 0.25 l min-1 to obtain a microaerobic atmosphere, and silicone water jackets maintained the temperature at 42 ÂºC. The flasks were placed on KMO 2 basic IKA-Werke stirrers (arbitrary setting, 260; Scientific Laboratory Supplies), which gaveeffective transfer of O2 from the gaseous atmosphere to the culture by virtue of a stable vortex. A fresh overflow sample was used to check the pH at the time of harvest (which was consistently between 6.6 and 7) and to ensure that there was no contamination by plating a 20 Î¼l sample onto both nutrient agar plates incubated at 37 Â°C at atmospheric oxygen, and Mueller-Hinton plates incubated at 42 Â°C in the microaerophilic work station. When staedy-state was reached, as verified by collecting at least 5 culture volumes, NOC-5 & -7 (final concentration 10 uM) was added to one culture. After a further 10 min the 125 ml volume was separated into 3 equal volumes and each was mixed immediately on ice with 3.56 ml 100% ethanol and 185 ml phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier. cDNA synthesis was carried out using 12 Âµg of starting material, which was primed with 9 Âµg of pd(N)6 random hexamers (Amersham Biosciences). Reaction mixtures (20 Âµl) containing 0.5 mM ATP, 0.5 mM TTP, 0.5 mM GTP, 0.2 mM CTP, and 1 mM Cy3- or 1 mM Cy5-dCTP were incubated for 2 h at 42 Â°C with 200 U of Superscript II RNase H reverse transcriptase (Invitrogen). Following synthesis, cDNA was purified by using a PCR purification kit (Qiagen) to remove the unincorporated deoxynucloside triphosphates, fluorescent dye, and primers. Equal volumes of cDNA were combined and evaporated to near dryness with an SPD121P Speed Vac (Thermon Savant). For hybridisation to the microarray slides, cDNA was resuspended in an appropriate volume of salt-based hybridization buffer (provided by Ocimum Biosolutions). Prior to addition to the slides, cDNA samples were heated to 95 Â°C for 3 min, and then placed on ice for no more than 3 min. The slides were placed in MWG Biotech hybridization chambers and incubated for 16 to 24 h in a shaking water bath at 110 rpm and 42 Â°C. Following incubation , the slides were washed sequentially for 5 min in 2x SSC-1 % sodium dodecyl sulphate, 1x SSC, 0.5x SSC and 0.1x SSC at 37 Â°C with gentle agitation (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The slides were dried by centrifugation at 254 xg for 5 min and subsequently scanned with an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained by using commercially available software from Biodiscovery, Inc. (Imagine version 4.0, and Genesight, version 3.5). The mean values from each channel were log2 transformed and normalized by using the subtract-by-mean method to remove intensity-dependent effects in the log2 (ratio) values. The Cy3/Cy5 fluorescence ratios were calculated from the normalized values. Biological experiments (i.e. chemostat growth with and without 0.25 mM GSNO addition) were carried out three times and a dye swap was performed for two of the three experiments which provided two technical repeats, one each for two of the three biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed â?¥ twofold and displayed and P value of â?¤ 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.

Dataset Information

Ciprofibrate stimulated gene expression in rats

Publications

Liver gene expression in rats in response to the peroxisome proliferator-activated receptor-alpha agonist ciprofibrate.

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