Expression Database

GUDMAP:12344

Theiler Stage

Supplemental Data Files

CEL file:	E15_5_MafB_3.CEL
CHP file:	E15_5_MafB_3_rma_gene_default.chp
RPT file:
TXT file:	E15_5_MafB_3.TXT

Archive ID

184

Principal Investigator(s)

Steve Potter, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA, steve.potter@cchmc.org

Submitted By

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

Specimen Details

Sample GEO ID:

GSM429048

Sample Description:

E15.5 Podocyte layer

Title:

E15_5_MafB_3

Component(s) Sampled:

visceral epithelium of capillary loop renal corpuscle (EMAP:27915) ; visceral epithelium of maturing renal corpuscle (EMAP:27969)

Organism:

Mus musculus

Strain:

CD-1

Genotype:

Non-wild type Allele:
Gene		Mafb
MGI ID		MGI:4847215
MGI Symbol or lab name		Tg(Mafb-EGFP)79Gsat
Type		transgenic insertion
Notes:		Mafb-EGFP-transgenic mice were received from the GENSAT Project an NIH-funded consortium that generates transgenic BAC-EGFP reporter lines.

Sex:

Unknown

Development Age:

15.5 dpc

Theiler Stage:

Pool Size:

approx 20 embryonic Kidneys

Pooled Sample:

Yes

Dissection Method:

Trypsinization & fluorescent activated cell sorting

Experimental Design:

MafB transgenic mice are time-mated. Pregnant MafB transgenic mice are euthanized by standard carbon dioxide asphyxiation. All fetuses are killed by decapitation with a scalpel. Fetal kidneys are dissected from fetuses and placed in ice-cold PBS. Embryonic kidneys are incubated in the presence of 300µl of trypsin for 5 minutes at 37 ºC. Kidneys are then dissociated by titurating in the presence of 600µl of ice-cold 10%FBS/PBS. The kidneys are pelleted at 5000 rpm, 4 ºC for 5 minutes. The media is aspirated and the cell pellet is resuspended in 200µl of ice-cold 2%FBS/PBS. Filter cells through 70 micron mesh filter and proceed with FACS.

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:	GSE17145
Number of Samples:	3 samples
Title:	Gene expression profiles of E15.5 developing podocytes in the developing kidney isolated from MafB-GFP transgenic mice using FACS on 1.0 ST Array chip. (GUDMAP Series ID: 33)
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:	MafB-GFP BAC transgenic mice were utilized to isolate the podocyte cells from the developing embryonic kidneys. Podocyte cells were isolated from the kidney using trypsinization and FACS. RNA was isolated 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