Unknown,Transcriptomics,Genomics,Proteomics

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Microarray analysis of gene expression of a Arabidopsis mutant hva-d

ABSTRACT: A microarray analysis was performed to compare the transcriptomes of the WT and the hva-d/+ heterozygous lines. All plants were grown under long-day conditions for five weeks. Three biological replicates were used for microarray analysis. Total RNA was isolated using an RNA isolation kit (Qiagen). The Affymetrix Gene Atlas System was used to perform the microarray analysis. The GeneAtlas® WT Expression Kit (Life Technologies Corp., Grand Island, NY, USA) was used for RNA treatment, cDNA synthesis, fragmentation and labeling. Background correction, quantile normalization and gene expression analysis were performed using the Transcriptome Analysis Console from Affymetrix. The gene-level differential expression filter was set at Fold Change (linear), and ANOVA P-value (Condition pair) < 0.05. Selected genes were then validated independently with real-time PCR experiments.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Huanzhong Wang

PROVIDER: E-MTAB-12850 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Pneumocystis is a pathogen of immunocompromised hosts but can also infect healthy hosts, in whom infection is rapidly controlled and cleared. To better understand the immune mechanisms contributing to clearance of infection, microarray methods were used to examine differential gene expression in the lungs of C57BL/6 and CD40 ligand knock-out (CD40L-KO) mice over time following exposure to Pneumocystis. Immuncompetent C57BL/6 mice, which control and clear infection efficiently, showed a robust response to infection characterized by the upregulation of 349 primarily immune-response associated genes. Temporal changes in the expression of these genes suggested that there was an early (week 2) primarily innate response, that waned without controlling infection; this were followed by primarily adaptive immune responses that peaked at week 5 and successfully cleared the infection. In conjunction with the latter, there was an increased expression of B cell associated (immunoglobulin) genes at week 6 that persisted through 11 weeks. In contrast, CD40L-KO mice, which are highly susceptible to developing severe Pneumocystis pneumonia, showed essentially no upregulation of immune-response associated genes at days 35 to 75. Immunohistochemical staining supported these observations by demonstrating an increase in CD4+, CD68+, and CD19+ cells in C57BL/6 but not CD40L-KO mice. Thus, the healthy host demonstrates a robust biphasic response to infection by Pneumocystis; CD40 ligand is an essential upstream regulator of the adaptive immune responses that efficiently control infection and prevent development of progressive pneumonia. Keywords: Time course response Pneumocystis murina infection wild type versus CD40L-KO mice In experiment 1, C57BL/6 and CD40L-KO mice were co-housed in 2 cages; one cage was exposed to a P. murina-infected seeder, while the second was unexposed. Mice (3 per group) were sacrificed at day 32. Because the initial study demonstrated very little change in gene expression in CD40L-KO mice, only C57BL/6 mice were used for experiments 2 and 3. In Experiment 2, three cages (10 animals/cage) were set up: 2 cages were exposed to P. murina, and one was unexposed. Animals (5 per cage) were sacrificed at days 34 and 41. In Experiment 3, which focused on gene expression early after exposure to P. murina, five cages (9-10 animals/cage) were set up, of which 3 were exposed and 2 were unexposed. Animals (1-3 per cage) were sacrificed at days 7, 14 and 21 of exposure. Experiment 4 was designed to verify the initial results in CD40L-KO mice, and included 5 unexposed controls and 4 mice exposed for 35 days.

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Microarray analysis of gene expression of a Arabidopsis mutant hva-d

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