|
| Status |
Public on Dec 23, 2014 |
| Title |
Control embryos replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
D.rerio_embryo_24hpf_control_3
|
| Organism |
Danio rerio |
| Characteristics |
developmental stage: Embryo (24 hpf)
groups: Control
|
| Treatment protocol |
Zebrafish embryos were injected at the single cell stage with 3.1 ng of zfglut2 translational start site blocking morpholino (5'-ACTGCTTCTCCATTTTGCATGAAGT-3') in 1× Danieau buffer (58mM NaCl, 0.7mM KCl, 0.4mM MgSO4, 0.6mM Ca(NO3)2, 5.0mM N-2-hydroxyethylpiperazine-N ́-2-ethanesulfonic acid; pH 7.6) containing 1% phenol red (Sigma-Aldrich). A standard control morpholino (5′-CCTCTTACCTCAGTTACAATTTATA-3′) (Gene Tools LLC) was used as negative control. At 72 hpf pools of 20 embryos per treatment group were snap-frozen in liquid nitrogen.
|
| Growth protocol |
Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (http://ZFIN.org). Embryos were grown at 28,5°C in egg water (60µg/ml Instant Ocean sea salts).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at -80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent (Invitrogen) and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI (Roche Applied Science) to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit (Qiagen) according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab-on-chip analysis using the 2100 Bioanalyzer (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
RNA was amplified and labeled with Cy3 dye using the single color Low Input Quick Amp Labeling kit (Agilent Technologies) following the manufacturer’s indications using 200 ng of RNA in each reaction. Next, 1,65 ng of labeled cRNA were hybridized to the arrays.
|
| |
|
| Hybridization protocol |
Overnight hybridization (17 hrs, 65ºC and 10 rpm rotation) was performed in a Microrarray Hybridization Oven (Agilent Technologies). After hybridization, microrarrays were washed with Gene Expression Wash Buffers 1 and 2 (Agilent Technologies).
|
| Scan protocol |
Microrarrays were scanned using the High-Resolution Scanner G2505C (Agilent Technologies).
|
| Description |
Control: Biological replicate 3
|
| Data processing |
Feature Extraction Software 10.7.3.1 (Agilent Technologies) was used for spot to grid alignment, feature extraction and quantification. Processed data were subsequently imported into GeneSpring GX 11.5 (Agilent Technologies).
|
| |
|
| Submission date |
May 20, 2014 |
| Last update date |
Dec 24, 2014 |
| Contact name |
Josep V Planas |
| E-mail(s) |
jplanas@ub.edu
|
| Phone |
+34-93-4039384
|
| Organization name |
Universitat de Barcelona
|
| Department |
Fisiologia i Immunologia (Biologia)
|
| Lab |
Fisiologia Molecular de Peixos
|
| Street address |
Av. Diagonal 643
|
| City |
Barcelona |
| ZIP/Postal code |
08028 |
| Country |
Spain |
| |
|
| Platform ID |
GPL13390 |
| Series (1) |
| GSE57836 |
Transcriptomic effects of GLUT2 knock-down in zebrafish embryos |
|
