Expression Database

GUDMAP:13523

Theiler Stage

Supplemental Data Files

CEL file:	Adult_Meis_3.CEL
CHP file:	Adult_Meis_3_rma_gene_default.chp
RPT file:
TXT file:	Adult_Meis_3.TXT

Archive ID

205

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:

GSM519448

Sample Description:

glomerular mesangium|extraglomerular mesangium

Title:

Adult_Meis_3

Component(s) Sampled:

glomerular mesangium (EMAP:29530) ; extraglomerular mesangium (EMAP:29534)

Organism:

Mus musculus

Strain:

CD-1

Genotype:

Non-wild type Allele:
Gene		Meis1
MGI ID		MGI:3843887
MGI Symbol or lab name		Tg(Meis1-EGFP)FO156Gsat
Type		transgenic insertion

Sex:

male

Development Age:

Adult

Theiler Stage:

Pool Size:

1 Adult kidney

Pooled Sample:

Dissection Method:

Collagenase A treatment, trypsinization & fluorescent activated cell sorting

Experimental Design:

Adult Meis BAC transgenic mice were euthanized by standard carbon dioxide asphyxiation. Kidneys were removed in ice-cold PBS, bisected and the medullary region removed using a dissecting microscope. The cortical region was minced into small pieces using a razor blade, then placed into a 1.5ml eppendorf tube containing 500µl of 0.5% Collagenase A. The tube was incubated at 37°C fro 10 minutes. Kidneys were then dissociated by titurating in the presence of 600µl of ice-cold 10%FBS/PBS. The cell suspension was filtered through a 100µM mesh to remove large cell debris. The flow-through was collected and refiltered using a 40µM mesh filter to capture glomeruli. The glomeruli were collected into 6cm petri dishes by overturning the 40µM mesh filter and rinsing the filter with ice-cold PBS. The purity of the glomeruli isolation was determined visually using a dissecting microscope. This process was repeated 2-3 times until pure glomeruli were isolated. The glomeruli were collected into a 1.5ml eppendorf tube and the tube was centrifuged at 2500 RPM for 5 min. The glomeruli were washed 2-3 times in ice-cold PBS. After the final rinse the PBS was removed and 300µl of 0.5% trypsin was added and incubated for 10-20 minutes. Every five minutes the glomeruli were triturated and were inspected under a microscope to visualize the amount of trypsinization. After incubation, the single-cell suspension was rinsed with 2%FBS/PBS three times. After the final wash, the cells were resuspended in 400µl of 1%FBS/PBS and refiltered through a 70µM mesh FACS tube.

Array Hybridization

Extracted Molecule:	Total RNA
A260:280 Ratio:
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:
Data Analysis Method:	Affymetrix Expression Console and GeneSpring 10
Reference:	PND 0.5 whole embryo RNA

Series Details

Series GEO ID:	GSE20687
Number of Samples:	3 samples
Title:	Gene expression profiles of adult mesangial cells isolated from Meis1-EGFP transgenic mice using FACS. (GUDMAP Series ID: 37)
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:	Meis1-EGFP BAC transgenic mice were utilized to isolate the mesangial cells from adult kidneys. The mesangial cells were isolated from the glomerulus using collagenase digestion, differential sieving, trypsin treatment and FACS. The RNA was isolated from purified mesangial 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