|
| Status |
Public on May 01, 2017 |
| Title |
Mutant normoxia 50 hpf |
| Sample type |
RNA |
| |
|
| Source name |
Zebrafish hif1α mutants, 50 hpf, normoxia
|
| Organism |
Danio rerio |
| Characteristics |
developmental stage: 50 hpf
sample type: 30 embryos
|
| Treatment protocol |
Two different conditions were considered: 50 hpf mutants and WT in normoxia, 50 hpf mutants and WT after hypoxia chamber treatment (2 hours after 3% O2)
|
| Growth protocol |
Zebrafish embryos were grown at 28 degree celsius in their medium
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using TRIZOL (Life Technologies) from 30 hif1α double mutants (hif1aabns89/bns89;hif1abbns90/bns90) and 30 WT siblings.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8 x 60K zebrafish expression array (XS-5090; Agilent 60-mer SurePrint technology)
|
| Description |
hif1α mutant at 50 hpf normoxia
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Oct 24, 2016 |
| Last update date |
May 01, 2017 |
| Contact name |
Claudia Gerri |
| E-mail(s) |
claudia.gerri@mpi-bn.mpg.de
|
| Organization name |
Max Planck Institute for Heart and Lung Research
|
| Department |
Developmental Genetics
|
| Lab |
Stainier
|
| Street address |
Ludwigstraße 43
|
| City |
Bad Nauheim |
| ZIP/Postal code |
61231 |
| Country |
Germany |
| |
|
| Platform ID |
GPL20686 |
| Series (1) |
| GSE89117 |
Gene expression signature of WT siblings and hif1α mutants in normoxia and after hypoxia chamber incubation |
|
