Comment[ArrayExpressAccession]	E-GEOD-25256
Public Release Date	2010-11-10
Investigation Title	The neural crest-enriched microRNA miR-452 regulates epithelial-mesenchymal signaling in the first pharyngeal arch									
Comment[Submitted Name]	The neural crest-enriched microRNA miR-452 regulates epithelial-mesenchymal signaling in the first pharyngeal arch
Experiment 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.									
Date of Experiment										
Term Source Name	EFO
Term Source Version										
Term Source File	http://efo.svn.sourceforge.net/viewvc/efo/trunk/src/efoinowl/efo.owl									
Person Last Name	Sheehy									
Person First Name	Neil									
Person Mid Initials										
Person Email	nsheehy@gladstone.ucsf.edu									
Person Affiliation	J. David Gladstone Institutes									
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Person Address	Gladstone Institute of Cardiovascular Disease, J. David Gladstone Institutes, 1650 Owens St., San Francisco, CA, USA									
Person Roles	submitter									
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Protocol Name	P-GSE25256-1	P-GSE25256-2	P-GSE25256-7	P-GSE25256-6	P-GSE25256-10	P-GSE25256-3	P-GSE25256-4	P-GSE25256-9	P-GSE25256-5	P-GSE25256-8
Protocol Description	ID_REF = <br>VALUE = Lowess normalization log2 ratio (Wnt1Cre,R26R YFP+)/(Wnt1Cre,R26R YFP-)	ID_REF = <br>VALUE = Lowess normalization log2 ratio (Dicerfl/+,Wnt1Cre,R26R)/(Dicer+/+,Wnt1Cre,R26R)	According to manufacturer's (Exiqon) recommendations	According to manufacturer's (Exiqon) recommendations	Equal number of Dicer heterozygotes and Dicer wildtypes were harvested at E10.5.  After removal from the embryonic sac, the region from just above the eyes to just below pharyngeal arch 6 was dissected out and placed in ice-cold PBS (Ca2+ and Mg2+ free).  The tissue was then sonicated to break up the tissue.  The tissue was then treated with 0.05% Trypsin/EDTA to generate single cells.  The cells were spun, resuspended in DMEM, and passed through a 40um nylon filter.  Cells from ten embryos were sorted into YFP+ populations and pooled for each genotype.	Equal number of Wnt1Cre,R26R YFP+ and Wnt1Cre,R26R YFP- were harvested at E11.5.  After removal from the embryonic sac, the region from just above the eyes to just below pharyngeal arch 6 was dissected out and placed in ice-cold PBS (Ca2+ and Mg2+ free).  The tissue was then sonicated to break up the tissue.  The tissue was then treated with 0.05% Trypsin/EDTA to generate single cells.  The cells were spun, resuspended in DMEM, and passed through a 40um nylon filter.   Cells from five E11.5 embryos were sorted into YFP+ and YFP- populations and pooled.	Total RNA was isolated from sorted cells using Trizol following the manufacturer's recommended protocols.	Equal number of Wnt1Cre,R26R YFP+ and Wnt1Cre,R26R YFP- were harvested at E10.5.  After removal from the embryonic sac, the region from just above the eyes to just below pharyngeal arch 6 was dissected out and placed in ice-cold PBS (Ca2+ and Mg2+ free).  The tissue was then sonicated to break up the tissue.  The tissue was then treated with 0.05% Trypsin/EDTA to generate single cells.  The cells were spun, resuspended in DMEM, and passed through a 40um nylon filter.  Cells from ten E10.5 embryos were sorted into YFP+ and YFP- populations and pooled.	Cy5 and Cy3 labeling protocol according to manufacturer's (Exiqon) recommendations	GPR files were read into R/Bioconductor using the m-array package. GenePix flagged spots were removed from subsequent analysis. Only good (unflagged) miR probes were used for normalization and subsequent analysis. M (log2 ratios) of Cy5 to Cy3 signals were calculated for each array (with NO background subtraction), and normalized by loess normalization. For each miRNA with more than 1 good probes, the median of normalized M of the replicate probes was taken as its summary value for each array.
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Protocol Type	bioassay_data_transformation	bioassay_data_transformation	image_aquisition	hybridization	nucleic_acid_extraction	nucleic_acid_extraction	nucleic_acid_extraction	nucleic_acid_extraction	labeling	feature_extraction
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Experimental Factor Name	TISSUE EXTRACTION	DEVELOPMENTAL STATE								
Experimental Factor Type	tissue extraction	developmental state								
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Comment[SecondaryAccession]	GSE25256									
Comment[GEOLastUpdateDate]	2010-11-10
Comment[AEExperimentType]	transcription profiling by array									
Comment[GEOReleaseDate]	2010-11-10
Comment[ArrayExpressSubmissionDate]	2010-11-10
SDRF File	E-GEOD-25256.sdrf.txt