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Omeprazole stimulated gene expression in rats

ABSTRACT: Omeprazole stimulated gene expression in rats Samples and RNA preparation: The series is composed of 9 hybridizations for analysis of differentially expressed genes in the gastric mucosa of omeprazole dosed vs. control rats. Nine rats (200-250 g) were given (by gavage) 400 micromol/kg of the proton pump inhibitor omeprazole (Astra AB, Gothenburg, Sweden) in Methocel (Dow Corning, Midland, MI) daily for 10 weeks. Seven rats received vehicle only. After 10 weeks the stomachs were removed for sampling of full-thickness corpus from the upper part of the greater curvature. Total RNA extraction was performed first by ultrasentrifugation on a cesium chloride cushion and then using TRIZOL Reagent (GIBCO BRL Life Technologies, New York, NY). Labeling and hybridization: Total RNA from 7 untreated rats were pooled and used as a common control. Three micrograms of total RNA from the control pool, and three micrograms of total RNA from each omeprazole dosed rat were reverse transcribed and labeled with capture sequences for subsequent attachment of Cy3- and Cy5-attached dendrimer, respectively, using 3DNA Array 350 Expression Array Detection kit as described in the manufacturer's protocols (Genisphere, Montvale, NJ). The labeling reactions were also spiked with exogenous control RNAs (Stratagene) at 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 slides were pre-washed in 2xSSC, 0.2% SDS at 55oC for 20 min, then 0.2x SSC at room temperature for 3 min, and deionized sterile filtered H2O at room temperature for 2 min. The Cy3 and Cy5 labeled samples (combined volume 9 microliter) were mixed with 25 microliter of the hybridization solution containing 0.25 M NaPO4, 1 mM EDTA, 4.5% SDS, 1 x SSC, 2 x Denhardt's solution and 2 microliter LNA dT Blocking reagent (Genisphere). A total hybridization volume of 50 microliter was added onto the array and covered with 24 x 60 mm cover slip. The array was assembled into humidified hybridization chamber (Corning) and hybridized at 60 oC for 18 h by submerging in a water bath. Post-hybridization washes were done once in 2 x SSC, 0.2% SDS at 55 oC 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. After washing the fluorescent 3DNA reagent was hybridized to the cDNA at 60 oC for 3 h as described in the manufacturer's protocols. After dendrimer hybridization the slides were washed as described above. Scanning, image analysis and data processing: Arrays were scanned at 10 micrometer resolution with a ScanArray Express HT scanner (Packard BioSciences, Billerica, MA). The microarrays were analyzed using GenePix™ Pro 4.1 (Axon Instruments, Inc., Union City, CA). All subsequent statistical analyses were performed using the statistical package R, R Development Core Team. A signal intensity based criterion was applied to remove the spots with median spot signal intensity less than 200. A spot area based criterion was applied to remove the spots with the area less than 50 pixels in either channel. Spots with more than 40% of the pixels saturated in either channel were also removed. Print tip-dependent normalization was applied to the data in order to compensate for systematic errors. The normalized ratios of the duplicated spots were averaged. Further analysis was done on the log2 transformed ratios. In order to assess the significance of up- or downregulation of the genes, tests for differential expression were performed using moderated T-tests, as implemented in the Limma R package of Smyth. Keywords: repeat sample

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE1808 | GEO | 2004/10/20

SECONDARY ACCESSION(S): PRJNA90289

REPOSITORIES: GEO

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Project description:Omeprazole stimulated gene expression in rats Samples and RNA preparation: The series is composed of 9 hybridizations for analysis of differentially expressed genes in the gastric mucosa of omeprazole dosed vs. control rats. Nine rats (200-250 g) were given (by gavage) 400 micromol/kg of the proton pump inhibitor omeprazole (Astra AB, Gothenburg, Sweden) in Methocel (Dow Corning, Midland, MI) daily for 10 weeks. Seven rats received vehicle only. After 10 weeks the stomachs were removed for sampling of full-thickness corpus from the upper part of the greater curvature. Total RNA extraction was performed first by ultrasentrifugation on a cesium chloride cushion and then using TRIZOL Reagent (GIBCO BRL Life Technologies, New York, NY). Labeling and hybridization: Total RNA from 7 untreated rats were pooled and used as a common control. Three micrograms of total RNA from the control pool, and three micrograms of total RNA from each omeprazole dosed rat were reverse transcribed and labeled with capture sequences for subsequent attachment of Cy3- and Cy5-attached dendrimer, respectively, using 3DNA Array 350 Expression Array Detection kit as described in the manufacturer's protocols (Genisphere, Montvale, NJ). The labeling reactions were also spiked with exogenous control RNAs (Stratagene) at 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 slides were pre-washed in 2xSSC, 0.2% SDS at 55oC for 20 min, then 0.2x SSC at room temperature for 3 min, and deionized sterile filtered H2O at room temperature for 2 min. The Cy3 and Cy5 labeled samples (combined volume 9 microliter) were mixed with 25 microliter of the hybridization solution containing 0.25 M NaPO4, 1 mM EDTA, 4.5% SDS, 1 x SSC, 2 x Denhardt's solution and 2 microliter LNA dT Blocking reagent (Genisphere). A total hybridization volume of 50 microliter was added onto the array and covered with 24 x 60 mm cover slip. The array was assembled into humidified hybridization chamber (Corning) and hybridized at 60 oC for 18 h by submerging in a water bath. Post-hybridization washes were done once in 2 x SSC, 0.2% SDS at 55 oC 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. After washing the fluorescent 3DNA reagent was hybridized to the cDNA at 60 oC for 3 h as described in the manufacturer's protocols. After dendrimer hybridization the slides were washed as described above. Scanning, image analysis and data processing: Arrays were scanned at 10 micrometer resolution with a ScanArray Express HT scanner (Packard BioSciences, Billerica, MA). The microarrays were analyzed using GenePixâ¢ Pro 4.1 (Axon Instruments, Inc., Union City, CA). All subsequent statistical analyses were performed using the statistical package R, R Development Core Team. A signal intensity based criterion was applied to remove the spots with median spot signal intensity less than 200. A spot area based criterion was applied to remove the spots with the area less than 50 pixels in either channel. Spots with more than 40% of the pixels saturated in either channel were also removed. Print tip-dependent normalization was applied to the data in order to compensate for systematic errors. The normalized ratios of the duplicated spots were averaged. Further analysis was done on the log2 transformed ratios. In order to assess the significance of up- or downregulation of the genes, tests for differential expression were performed using moderated T-tests, as implemented in the Limma R package of Smyth.

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Project description:Synechocystis 6803 cells was grown photoautotrophically at 32 °C buffered in BG-11 and bubbled with 3% CO2. A relatively mild Ci stress was applied by switching the aeration from 3% CO2 to air alone. After incubation under designated conditions, a 100-ml aliquot of culture was immediately combined with an equal volume of ice-cold mixture of phenol and ethanol (1:10, w/v) in an ice bath. The resultant cells were collected by centrifugation at 1000 x g for 10 min at 4 °C. Total RNA was isolated with RNeasy Midi Kit (Qiagen, Valencia, CA) and further treated with the DNA-free kit (Ambion, Austin, TX). Fluorescently labeled cDNA was produced via a two-step indirect procedure involving cDNA synthesis from 16 µg of total RNA in a reverse transcriptase reaction incorporating aminoallyl-modified deoxynucleotide, followed by the second step involving chemical coupling of fluorescent dye to the introduced amino moieties of the synthesized cDNA. Labeled cDNA were adjusted to 14.75 µl, and the remainder of the hybridization components containing 2.5 µl of 10 µg µl-1 salmon sperm DNA, 8.75 µl of 20x SSC, 0.25 µl of 10% SDS, and 8.75 µl of formamide were added. The mixture was then heated for 2 min at 99 °C and maintained at 42 °C until hybridization. Hybridizations were preformed in a static incubator at 42 °C for 12-16 h then washed by placing in a 250-ml solution of 2x SSC and 0.1% SDS at 42 °C for 5 min with gentle agitation provided by rotation of a magnetic stir bar. The slide was transferred quickly to a 250-ml solution of 0.1x SSC and 0.1% SDS, incubated for 10 min at room temperature with gentle agitation, and washed five additional times in 0.1x SSC for 1 min at room temperature. Hybridization signals from the microarray were quantified using GenePix Pro 4.1 (Axon Instruments, Union City, CA). The quality control procedures were conducted in the image analysis software, and then data were saved to Acuity 3.1 (Axon Instruments). Keywords: time-course

Project description:This series repressents the data set from the paper "Expression microarray analysis and oligo array CGH of aquired gemcitabine resistance in mouse colon reveals selection for chromosomal aberrations". Biological material: One solid mouse tumor "Colon 26A" was routinely maintained by successive transplantation. A subset of mice with this tumor was treated at the maximum tolerable dosage of Gemcitabine and successively transplanted 5 times and similarly treated. The last passage was completly resistant and designated, "Colon 26G". Expression array: Total RNA was isolated from frozen mouse tumors using Tryzol. Single stranded cDNA was synthetized from 30 mg of total RNA by reverse transcription using aminoallyl labeled dUTP (Ambion). Labelling was performed according to the aminoallyl labeling protocol. Briefly, cDNA was incubated at room temperature for 1 h with fluorolink monofunctional cy5 or cy3 (Amersham) followed by 15 min 4 M hydroxylamine treatment. Uncoupled dyes were removed using Qiaquick PCR purification columns (Qiagen) and mixed with 12 mg poly(dA) (Amersham), 60 mg yeast tRNA (Sigma) and 24 mg Cot-1 DNA (Invitroen). The probe was dissolved in 127 ml hybridization mixture containing 46% formamide, 9.5% dextran sulfate, 2x SSC and 0.2% SDS. The probe was heated to 70°C for 10 min and annealed at 37°C for 1 h. Slides were hybridized for 14 h at 37°C over-night (HyArray 12™, Perkin Elmer). After hybridization the slides were washed in the HybArray 12™ with 50% formamide, 2x SSC, pH 7 at 35°C for 15 min, followed b PI buffer (0.1 M sodium phosphate, 0.1% Igepal Ca630, pH 8) at room temperature and 3 washes of 0.2x SSC, 0.1x SSC and 0.01x SSC at room temperature followed by centrifugation. Arrays were scanned using a laser scanner (ScanArray Express, Perkin Elmer) and analysed using ImaGene version 5.6. Cy5 cy3 ratios are calculating by taking the log2 of the signal mean of each spot minus the median background mean of all spots on the array. This is followed by a standard normalization for spot intensity and calculation of the ratios. Keywords: other

Project description:MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon head kidney. Head kidney mRNA samples were prepared from pooled (n = 10) infected or control kidney. Approximately 200 ng of infected or control head kidney mRNA was used as template in aRNA synthesis reactions. Two infected head kidney samples were pooled to yield 8.9 ug of aRNA, and a single control head kidney sample contained 8.4 ug of aRNA. Infected and control head kidney aRNA samples were visualized on agarose gels to check quality and quantity (data not shown). Except for the amounts of aRNA and reverse transcription (RT) primer used, head kidney target labeling reactions and microarray hybridizations were identical. Head kidney target syntheses used 2 ug of aRNA and 2 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C for 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5’T18[Q-N]3’), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before. Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 75 Cy3, PMT 63 or 64 Cy5 for head kidney study). Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery). Keywords: other

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Project description:MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon macrophages. Infected and control (24 h incubation) macrophage mRNA samples were subjected to two rounds of amplification using the MessageAmp aRNA kit (Ambion) and manufacturer's instructions. Approximately 400 ng of macrophage mRNA was used as template in the first round of amplification, yielding 8.1 ug of infected macrophage aRNA and 7.8 ug of control macrophage aRNA. Approximately 1 ug of first-round macrophage aRNA was used as template in the second round of amplification, yielding 81.2 ug of infected macrophage aRNA and 83.3 ug of control macrophage aRNA. Infected and control macrophage aRNA samples were visualized on agarose gels to check quality and quantity. Except for the amounts of aRNA and reverse transcription (RT) primer used, macrophage target labeling reactions and microarray hybridizations were identical. Macrophage target syntheses used 5 ug of second-round aRNA and 5 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5'T18[Q-N]3'), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before. Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 72 Cy3, PMT 63 or 64 Cy5 for macrophage study). Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery). Keywords: other

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Omeprazole stimulated gene expression in rats

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