Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiling of Wt1 mutant renal anlagen

ABSTRACT: Wt1 is required for renal development and homozyogus knockout mice show renal agenesis caused by apoptosis of the metanepric mesnchyme. To identify genes regulated by WT1, we performed comparative gene expression profiling on kidney rudmients of Wt1 heterozygous and homozygous mutant E10.25 embryos. To identify deregulated genes, timed matings were performed and embryos isolated at E10.25, a time point before the onset of apoptosis in Wt1 mutants. The region containing the metanephric mesenchyme was microdissected, RNA isolated and after labeling, hybridized to Mouse Genome Survey Microarrays. Deregulated genes were identified using GeneSpring (GSX) software.

ORGANISM(S): Mus musculus

SUBMITTER: Andreas Schedl

PROVIDER: E-GEOD-58073 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Microarray experiment on each time point and genotype was performed in technical duplicates (ie. a single RNA preparation of pooled kidneys of one genotype used for two separate target preparations). Data from a total of 8 independent arrays were used in this study, 2 arrays were used for RNA samples from E18.5 control kidneys, 2 for E18.5 Lim1 conditional mutant kidneys, 2 for E14.5 control kidneys, and the other 2 for E14.5 Lim1 conditional mutant kidneys. Data generated from all arrays that satisfied the preliminary analysis were exported and loaded into DNA-Chip Analyzer (dChip2004), where statistical and comparative analyses were performed to verify the data. The data were normalized using the default normalization method. Briefly, an iterative procedure was used to identify an invariant set of probes, which presumably consisted of non-differentially expressed genes. A piecewise-linear running median curve was then calculated and used as the normalization curve. After normalization, all arrays had similar brightness. Median intensities around 155 (between 155 to 158) were obtained after normalization. Percent gene present (P call%) values between 55.7% and 63.6% were observed using default detection p-value cut offs (a1=0.04 and a2=0.06). Array outlier (%) and single outlier (%) were detected at ranges from 0.016% to 0.080% and from 0.009% to 0.045%. Expression data obtained from all arrays used are provided. Normalized data were exported in a tab delimited text format. Fold changes of each transcript from different samples were calculated and sorted using Microsoft Excel 5.0 software. Signal obtained from control kidney samples were used as an experiment to compare to the signal obtained from Lim1 conditional mutant kidneys that was designated as a baseline. A 2-fold change in the means of signal obtained from experimental duplicates and those from baseline duplicates was used as the criterion to identify differentially expressed transcripts. To ensure the quality of the data, probe sets that showed a fold change between duplicates greater than between the experimental mean and baseline mean were removed. To study only genes that showed consistent expression on experimental chips, probe sets that did not show consistent present calls in the experimental duplicates were removed. To focus on genes with a significant fold change between the experiment and the baseline, only probe sets that the product of their experimental mean and fold change were more than 100 were retained. To produce a compact differentially expressed gene list, the probe set list was sorted within Microsoft Excel based on Locus Link number and redundant entries were removed. Our experimental design description and the data format provided in the Additional files fulfill the MIAME (minimum information about a microarray experiment) standards.

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Gene expression profiling of Wt1 mutant renal anlagen

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