|
| Status |
Public on Jul 16, 2010 |
| Title |
Adult_MafB_3 |
| Sample type |
RNA |
| |
|
| Source name |
visceral epithelium (syn: podocyte layer)
|
| Organism |
Mus musculus |
| Characteristics |
gene reported: Tg(Mafb-EGFP)79Gsat / MMRRC:011834-UCD
strain: CD-1
sex: Male
developmental stage: Adult
theiler stage: TS28
somite count: NA
developmental landmark: NA
|
| Treatment protocol |
pool size: approx 2 Adult Kidney
Pooled sample: Yes
Dissection Method: Podocyte cells were isolated from adult Mafb transgenic mice kidneys using a combination of Collagenase A digestion, sieving and tyrpsinization followed by FACS.
7- to 9-week-old Mafb transgenic adult male mice were euthanized by standard carbon dioxide asphyxiation. The kidneys were isolated and placed in ice-cold PBS. The kidneys were minced with a razor blade and incubated in the presence of 300 µl of 0.5% Collagenase A for 15 minutes at 37ºC. After incubation, the kidneys were further dissociated by titurating in the presence of 600 µl of ice-cold 10% FBS/PBS, and then pelleted at 5000 rpm, 4ºC for 5 minutes. The cells were resuspended in 500 µl of ice-cold 2% FBS/PBS. Glomeruli were isolated using a decreasing series of filter sizes (100µm, 70 µm, 40 µm). The purified glomeruli were then incubated with 0.5% trypsin-EDTA for 5-10 minutes at 37ºC. The cells were then washed several times with 500 µl of ice-cold 2% FBS/PBS, filtered using a 70 µm mesh. The GFP positive podocytes were isolated using FACS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Potter protocol: 'RNA purification'
|
| Label |
Biotin
|
| Label protocol |
The FL-Ovation cDNA Biotin Module V2 (NuGEN) is used to chemically modify fragments and label target sense cDNA.
|
| |
|
| Hybridization protocol |
Affymetrix standard protocol.
Amount labeled target hybridized to array: 2.5µg
The arrays were washed and stained using a Fluidics Station 450 (Affymetrix) utilizing 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.
|
| Description |
>> Amplification protocol <<
Target Amplified manufacturer/kit: WT-Ovation Pico RNA Amplification System (NuGEN)
Target Amplified protocol: Potter protocols
Rounds of amplification: 1
URL: http://www.gudmap.org/gudmap/pages/mic_submission.html?id=GUDMAP:12338
|
| Data processing |
Analysis method: Affymetrix Expression Console and GeneSpring 10.
|
| |
|
| Submission date |
Jul 16, 2009 |
| Last update date |
Dec 31, 2010 |
| Contact name |
GUDMAP Developers |
| E-mail(s) |
gudmap-db@gudmap.org
|
| Phone |
+44 131 651 8500
|
| Organization name |
IGMM MRC Human Genetics Unit
|
| Lab |
GUDMAP Database Group
|
| Street address |
Crewe Road
|
| City |
Edinburgh |
| ZIP/Postal code |
EH4 2XU |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL6246 |
| Series (2) |
| GSE17142 |
Gene expression profiles of adult visceral epithelium (syn: podocyte layer) isolated from MafB-GFP transgenic mice using FACS. (GUDMAP Series ID: 31) |
| GSE17218 |
Encyclopedia of the expression levels of all genes in multiple components of the developing kidney |
|
