|
| Status |
Public on Oct 19, 2017 |
| Title |
wt_Ahsg_1 |
| Sample type |
RNA |
| |
|
| Source name |
growth plate cartilage from wildtype mouse
|
| Organism |
Mus musculus |
| Characteristics |
tissue: growth plate cartilage
age: 13 days postnatal
Sex: male
genetic background: C57BL/6n
genotype: Ahsg+/+
|
| Extracted molecule |
total RNA |
| Extraction protocol |
From each mouse, the right femur was directly frozen in OCT in isopentane cooled with dry ice. Cryo-section preparation was performed under nuclease-free conditions to maintain RNA integrity. Sections of 14 µm were mounted on PEN-membrane slides (Zeiss). Slides were fixed with 95 % ethanol for 2 min, dehydrated with 100 % ethanol for 1 min, air dried at room temperature for 5 min and stored at 80 °C for up to 48 h until further processing. Laser capture microdissection (LCM) was performed using a Zeiss P.A.L.M. micro laser system. For each sample, approx. 40 frozen sections were prepared and processed. Per section, the entire growth plate (resting zone, proliferative zone and hypertrophic zone) was isolated and captured in an adhesive cap (Zeiss). The isolated tissue was suspended in 50 µl extraction buffer (PicoPure RNA Isolation Kit, Thermo Fisher Scientific), incubated at 42 °C for 20 min and stored at 80 °C until RNA isolation, which was carried out according to the manufacturer’s protocol.
|
| Label |
biotin
|
| Label protocol |
Fragmented, biotinylated cDNAs were prepared from 150 ng total RNA using the Affymetrix WT plus reagent kit according to the manufacturers instructions.
|
| |
|
| Hybridization protocol |
Samples were hybridized using Affymetrix haybridization kit materials according to the manufacturers instructions: hybridization 45°C for 16 hours at 60 rpm; Fluidics Station Program FS_450_0001
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G.
|
| Data processing |
Raw image data were analysed with Affymetrix® Expression Console™ Software (Affymetrix, USA), and gene expression intensities were normalized and summarized with SST robust multiarray average algorithm (Affymetrix): SST-RMA. Further analyses was performed using the Transcriptome Analysis Console (TAC) Software of Affymetrix.
|
| |
|
| Submission date |
Oct 18, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Bernd Denecke |
| E-mail(s) |
[email protected]
|
| Phone |
+49 241 8089918
|
| Organization name |
RWTH Aachen University
|
| Department |
IZKF Aachen
|
| Street address |
Pauwelsstrasse 30
|
| City |
Aachen |
| State/province |
NRW |
| ZIP/Postal code |
52074 |
| Country |
Germany |
| |
|
| Platform ID |
GPL20258 |
| Series (1) |
| GSE105139 |
Post-weaning epiphysiolysis causes distal femur dysplasia and foreshortened hindlimbs in fetuin-A-deficient mice |
|
