|
| Status |
Public on Jul 16, 2010 |
| Title |
Remec_PO3 |
| Sample type |
RNA |
| |
|
| Source name |
juxtaglomerular complex
|
| Organism |
Mus musculus |
| Characteristics |
gene reported: Ren1c-YFP / PMID:18055510
strain: CD-1
Sex: unknown
developmental stage: E18.5
theiler stage: TS26
somite count: NA
developmental landmark: NA
|
| Treatment protocol |
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.
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.
|
| 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:12329
|
| Data processing |
Analysis method: Affymetrix Expression Console and GeneSpring 10.
|
| |
|
| Submission date |
Jul 16, 2009 |
| Last update date |
Feb 21, 2012 |
| 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) |
| GSE17138 |
Gene expression profiles of renin producing cells in newborn and adult kidney isolated from Renin-YFP transgenic mice using FACS. (GUDMAP Series ID: 29) |
| GSE17218 |
Encyclopedia of the expression levels of all genes in multiple components of the developing kidney |
|
