Expression Database

GUDMAP:12329

Theiler Stage

Images

Supplemental Data Files

CEL file:	Remec_PO3.CEL
CHP file:	Remec_PO3_rma_gene_default.chp
RPT file:	NA
TXT file:	Remec_PO3.TXT

Archive ID

161

Principal Investigator(s)

Steve Potter, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA, steve.potter@cchmc.org
R Ariel Gomez, University of Virginia, Charlottesville, VA, USA, rg@hscmail.mcc.virginia.edu

Submitted By

Eric Brunskill, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA, brune0@cchmc.org

Specimen Details

Sample GEO ID:

GSM429016

Sample Description:

P0_developing_JGA

Title:

Remec_PO3

Component(s) Sampled:

juxtaglomerular complex (EMAP:30452)

Organism:

Mus musculus

Strain:

CD-1

Genotype:

Non-wild type Allele:
Gene		Ren1
MGI Symbol or lab name		Ren1c-YFP
Type		transgenic insertion
Notes:		Transgenic mouse obtained from Dr R Ariel Gomez, University of Virginia. See PMID:18055510.

Sex:

Unknown

Development Age:

18.5 dpc

Theiler Stage:

Pool Size:

approx 4 E18.5 kidneys

Pooled Sample:

Yes

Dissection Method:

Juxtaglomerular cells from Ren1c-YFP transgenic mice kidneys were isolated using a combination of Collagenase A digestion and sieving followed by FACS.

Experimental Design:

Renin expressing cells were isolated by FACS from kidneys of our Ren1c-YFP mice (Pentz et al., 2008) in which renin expression is marked by YFP. The kidneys were dissected out in ice-cold PBS and the kidney and isolated cells were kept cold at all times. The medulla was removed and the cortex was digested with collagenase A, washed and then digested with trypsin. After stopping the trypsin digestion, the cells were washed and filtered through a 100µm filter. Red blood cells were lysed, the cells were washed and then filtered through a 70µm filter. Cells were pelleted, resuspended in 2%FBS/PBS and sorted immediately using a high-speed digital BD FACS Aria II Cell Sorter. As a control, cells were isolated in parallel from wild type kidneys to provide the baseline for excluding unlabeled cells.

Array Hybridization

Extracted Molecule:	Total RNA
A260:280 Ratio:	NA
RNA Extraction Protocol:	Potter protocol: 'RNA purification'
Target Amplification Manufacturer/kit:	WT-Ovation Pico RNA Amplification System (NuGEN)
Target Amplification Protocol:	Potter protocols
Rounds of Amplification:	1
Amount Labeled Target Hybridized To Array:	2.5µg
Label:	Biotin
Label Protocol:	The FL-Ovation cDNA Biotin Module V2 (NuGEN)
Array Hyb/Wash Protocol:	Affymetrix Standard Protocol. The arrays were washed and stained using a Fluidics Station 450 (Affymetrix) utilising the fluidics protocol FS450-0007.
Scan Protocol:	Probe arrays were scanned using an Affymetrix GeneChip Scanner 3000 7G and Affymetrix scanning software Genechip Operating Software Version 1.4.
GCOS Tgt Value:	NA
Data Analysis Method:	Affymetrix Expression Console and GeneSpring 10
Reference:	PND 0.5 whole embryo RNA

Series Details

Series GEO ID:	GSE17138
Number of Samples:	6 samples
Title:	Gene expression profiles of renin producing cells in newborn and adult kidney isolated from Renin-YFP transgenic mice using FACS. (GUDMAP Series ID: 29)
Summary:	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
Type:	Expression profiling of kidney components by microarray
Overall Design:	Renin-YFP transgenic mice were utilized to isolate renin producing cells from the kidneys of either P0 neonatal pups or Adult mice. Renin producing cells were isolated from the kidney by fluorescent activated cell sorting (FACS). RNA was isolated from FACS sorted cells and the gene expression profiles were determined by microarrays.

Platform Details

Platform GEO ID:	GPL6246
Title:	Affymetrix Mouse Gene 1.0 ST Array
Distribution:	commercial
Technology:	in situ oligonucleotide
Organism:	Mus musculus
Manufacturer:	Affymetrix
Manufacturer Protocol:	N/A
Catalogue Number:	N/A