Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Gene expression profiles of E11.5 metanephric mesenchyme and Adult Juxtaglomerular Complex cells or renal cortex from the kidneys of Crym-GFP or Ren1c-YFP transgenic mice using FACS or single-cell isolation techniques (GUDMAP Series ID:47).

ABSTRACT: The long term objective is to create an encyclopedia of the expression levels of all genes in multiple components of the developing kidney. The central thesis is straightforward. The combination of fluorescent activated cell sorting (FACS) plus microarray analysis offers a powerful, efficient and effective method for the creation of a global gene expression atlas of the developing kidney. Microarrays with essentially complete genome coverage can be used to quantitate expression levels of every gene in FACS isolated components of the developing kidney. The ensuing rapid read-out provides an expression atlas that is more sensitive, more economical and more complete than would be possible by in situ hybridizations alone. YFP & GFP transgenic lines have been used to isolate several cell types including the metanephric mesenchyme, Juxtaglomerular Complex cells or renal cortex from the kidneys of either E11.5 embryos or adult mice. The various cell types were isolated from the kidney using microdissection and single-cell isolation techniques. RNA was isolated from cells and the gene expression profiles were determined by microarrays.

ORGANISM(S): Mus musculus

SUBMITTER: GUDMAP Developers

PROVIDER: E-GEOD-42713 | 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.

Project description:S23 experiment: We sought to identify the microRNAs (miRNAs) enriched in the neural crest cell (NCC) population from E11.5 mouse embryos. To accomplish this, we utilized a transgenic mouse line harboring Cre-recombinase under the control of the Wnt1 NCC-specific promoter and also carrying the R26R-YFP allele. We sorted YFP+ (NCCs) and YFP- (non-NCCs) from E11.5 Wnt1Cre-R26R mouse embryos via FACS and compared the relative enrichment of miRNAs in the YFP+ population by miRNA microarray. 220 experiment: We sought to identify the microRNAs (miRNAs) enriched in the neural crest cell (NCC) population from E10.5 mouse embryos. To accomplish this, we utilized a transgenic mouse line harboring Cre-recombinase under the control of the Wnt1 NCC-specific promoter and also carrying the R26R-YFP allele. We sorted YFP+ (NCCs) and YFP- (non-NCCs) from E10.5 Wnt1Cre-R26R mouse embryos via FACS and compared the relative enrichment of miRNAs in the YFP+ population by miRNA microarray. 221 experiment: Our research has shown that heterozygous deletion of the miRNA processing enzyme Dicer leads to developmental delay of the thymus in mouse embryos. We sought to identify the microRNAs (miRNAs) affected by the loss of a single copy of Dicer in the neural crest cell (NCC) population from E10.5 mouse embryos. To accomplish this, we utilized a transgenic mouse line harboring a floxed allele of Dicer, Cre-recombinase under the control of the Wnt1 NCC-specific promoter, and also carrying the R26R-YFP allele. We sorted YFP+ (NCCs) cells from E10.5 Dicerfl/+,Wnt1Cre,R26R and Dicer+/+,Wnt1Cre,R26R mouse embryos via FACS and compared the relative expression of miRNAs in the Dicer-heterozygotes compared to Dicer-wildtypes by miRNA microarray. S23 experiment: RNA from YFP+ (NCCs) and YFP- (non-NCCs) cells sorted by FACS from E11.5 Wnt1Cre-R26R mouse embryos was isolated and hybridized to Exiqon miRNA microarrays v10. Cells from five E11.5 embryos were sorted into YFP+ and YFP- populations and pooled. 220 experiment: RNA from YFP+ (NCCs) and YFP- (non-NCCs) cells sorted by FACS from E10.5 Wnt1Cre-R26R mouse embryos was isolated and hybridized to Exiqon miRNA microarrays v10. Cells from ten E10.5 embryos were sorted into YFP+ and YFP- populations and pooled. 221 experiment: RNA from YFP+ (NCCs) cells sorted by FACS from E10.5 Dicerfl/+,Wnt1Cre,R26R and Dicer+/+,Wnt1Cre,R26R mouse embryos was isolated and hybridized to Exiqon miRNA microarrays v10. Cells from ten embryos were sorted into YFP+ populations and pooled for each genotype.

Dataset Information

Gene expression profiles of E11.5 metanephric mesenchyme and Adult Juxtaglomerular Complex cells or renal cortex from the kidneys of Crym-GFP or Ren1c-YFP transgenic mice using FACS or single-cell isolation techniques (GUDMAP Series ID:47).

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