实验库 数据相关信息
- 题目:
- Omeprazole stimulated gene expression in rats
- ID:
- 状态:
- 发布时间June 5, 2010 , 更新时间 March 27, 2012 , 提交时间 Sept. 28, 2004,
- 物种:
- Rattus norvegicus
- 摘要:
- 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.
- 实验种类:
- unknown experiment type
- 样本量:
- 18
- 实验设计:
- 无设计数据
- 数据号:
- E-GEOD-1808, GSE1808
- 数据状态:
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DataTable pData
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