Reproducibility of global gene expression in rat lung
Ontology highlight
ABSTRACT:
ORGANISM(S): Rattus norvegicus
SUBMITTER: Satoru Tsunemine
PROVIDER: E-GEOD-36109 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:We used microarrays to assess the reproducibility of global gene expression in rat lung from experiments performated by 2 individuals. Expression analyses in 01matsu and 01suzu rat lung. Both prepared under the same conditions, and the comparison is between the reproducibility of the experiment as performed by two different individuals.
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Project description:The study sought to investigate differences in onset and progression of renal disease in the Dahl salt-sensitive (S), S.SHR(2) congenic, and spontaneously hypertensive rat (SHR) using a time-course. The data clearly demonstrates that the locus on chromosome 2 has a major and sustained ability to attenuate renal damage. As early as week 4, significant interstitial changes were observed between the S and the congenic which preceded any significant difference in proteinuria. Gene expression profiling was performed at week 4, 12, and 20 using kidney from the S and S.SHR(2) congenic to: (1) identify expression differences of positional candidates within the QTL region; (2) correlate temporal gene expression changes between the S and congenic with degree of renal damage; and (3) identify biochemical pathways potentially involved in the attenuated renal damaged observed in the congenic. Gene pathway analysis (Ingenuity® Systems) performed at week 4, 12, and 20 revealed that pathways involved in cellular assembly and organization, cellular movement, and immune response were controlled differently between the S and congenic. Considering all the data, the chromosome 2 congenic appears to attenuate renal damage primarily through an altered fibrotic response. Experiment Overall Design: DNA microarray analysis was performed using Affymetrix Genechip® Rat Genome 230 2.0 array at three timepoints. The chip contains 31,000 probe sets from more than 30,000 transcripts on one array. Three male S and three male S.SHR(2) congenic were selected at random from each group test for proteinruia at week 4, 12 and 20. Kidney was cut into ~ 0.5cM size cubes, suspended in RNAlater (Ambion, Austin, TX) and stored overnight at 4ºC. RNA was extracted using Trizol® reagent (Invitrogen, Carlsbad, CA) and purified using Mini RNeasy kit (Qiagen,Valencia, CA ) according to manufacturerâs protocols. RNA quality was assessed by an OD260/280 ratio > 2.0 and visually by ethidium bromide staining on an agarose gel. Experiment Overall Design: Biotinylated cRNA was synthesized from 10 ug of total RNA using the One-Cycle Target Labeling Kit (Affymetrix, Santa Clara, CA) as directed by the user manual. cRNA quality for each sample was assessed by hybridization of 5ug adjusted kidney cRNA to Affymetrix Genechip® Test3 array. Subsequently, 15ug adjusted kidney cRNA was hybridized to the Genechip® Rat 230 2.0 array. Hybridized chips were automatically washed, stained and scanned at the Medical University of Ohio Bioinformatics & Proteomics/Genomics Program gene array facility using Affymetrix equipment.
2008-06-14 | E-GEOD-6208 | biostudies-arrayexpress
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Project description:Left heart disease (LHD) frequently causes lung vascular remodelling and pulmonary hypertension (PH). Yet, pharmacological treatment for PH in LHD is lacking and its pathophysiological basis remains obscure. We aimed to identify candidate mechanisms of PH in LHD and to test their relevance and therapeutic potential. In rats, LHD was induced by supracoronary aortic banding. Whole genome microarray analyses were performed, candidate genes were confirmed by RT-PCR and Western blots and functional relevance was tested in vivo by genetic and pharmacological strategies. In lungs of LHD rats, mast cell activation was the most prominently upregulated gene ontology cluster. Mast cell gene upregulation was confirmed at RNA and protein levels and remodelled vessels showed perivascular mast cell accumulations. In LHD rats treated with the mast cell stabiliser ketotifen, or in mast cell deficient Ws/Ws rats, PH and vascular remodelling were largely attenuated. Both strategies also reduced PH and vascular remodelling in monocrotaline-induced pulmonary arterial hypertension, suggesting that the role of mast cells extends to noncardiogenic PH. In PH of different aetiologies, mast cells accumulate around pulmonary blood vessels and contribute to vascular remodelling and PH. Mast cells and mast cell-derived mediators may present promising targets for the treatment of PH. Whole rat genome microarray analyses were performed in lung homogenates of three rats with established PH following supracoronary aortic banding and in three sham-operated controls. Out of a total of 28,000 analysed genes, differential expression defined as 2-fold change with p<0.05 was evident for 120 genes. Of these, 76 were upregulated and 44 downregulated in aortic banding compared with control lungs gene. Enrichment analysis revealed regulation of mast cell specific genes - 13 out of 20 genes were significantly upregulated in aortic banding compared with control lungs. To test for a putative functional role of mast cells in lung vascular remodelling and PH in LHD, we applied a pharmacological approach by treatment of aortic banding rats with the mast cell stabiliser ketotifen. Microarray analysis then compared rats that were treated with untreated. In brief, congestive heart failure (CHF) was induced in juvenile rats by supracoronary aortic banding and rats were analyzed 9 weeks thereafter when they had established left heart failure with preserved ejection fraction and secondary pulmonary hypertension. Three heart failure rats were untreated, and 3 received the mast cell stabilizer ketotifen (1 mg/kg-1 bodyweight/day-1) with the drinking water. Sham rats underwent the same surgical procedure but without placement of a clip on the supracoronary aorta. Microarray analyses: Lungs from banded and control rats were excised and total RNA was extracted (Stratagene Absolutely RNA Miniprep Kit; Stratagene, La Jolla, USA). Three µg total RNA from three control and three banded rats each were processed according to the One-Cycle Target Labeling protocol (GeneChip Expression Analysis, Affymetrix, Santa Clara, USA). Before and after amplifications, the total RNA/complementary RNA concentrations were checked with NanoDrop ND-1000 (Thermo Scientific, Wilmington, USA) and quality was controlled (Experion electrophoresis station, BioRad; Hercules, USA). Samples were hybridized to GeneChip® Rat Genome 230 2.0 arrays (Affymetrix; Santa Clara, USA) containing 31,000 probe sets covering 28,000 rat genes.
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