|
| Status |
Public on Dec 14, 2013 |
| Title |
Pineal_Dark_CT2 |
| Sample type |
RNA |
| |
|
| Source name |
pineal glands at CT2, constant darkness
|
| Organism |
Danio rerio |
| Characteristics |
treatment: constant darkness
time point: CT2
tissue: pineal gland
gender: males and females
age: adult (0.5-1.5 years old)
strain: Tg(aanat2:EGFP)Y8 transgenic zebrafish
|
| Treatment protocol |
Fish were transferred to constant darkness (DD) at the end of the day prior to the experiment. Fish were exposed to a 1-hr light pulse (light intensity of 12 W/m2) prior to sampling (light treatment) or kept under constant darkness for control (dark treatment). The tissues were collected from light- and dark-treated fish at 6 time points with 4-hr intervals throughout one daily cycle, corresponding to CT2, 6, 10, 14, 18 and 22. Fish were anesthetized in 1.5 mM Tricane (Sigma) and sacrificed by decapitation, and pineal glands were removed under a fluorescent dissecting microscope. 12 pineal glands were collected and pooled at each time point.
|
| Growth protocol |
Adult zebrafish were raised in a light- and temperature-controlled recirculation water system under a 12-h light/12-h dark cycle at 28 °C and fed twice a day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Extraction of total RNA was performed according to the manufacturer's instructions (RNeasy, Qiagen).
|
| Label |
biotin
|
| Label protocol |
Double-strand cDNA was generated from 10-100 ng of mRNA by using a T7-linked oligo(dT) primer (Affymetrix). After second-strand synthesis, in vitro transcription was performed with MEGAscript® T7 Kit (Ambion, Inc.). The cRNA (600ng) was then used for second cycle first-strand cDNA (Affymetrix) by using a T7-linked oligo(dT) primer (Affymetrix). After 2nd cycle - second-strand synthesis, in vitro transcription was again performed; this time biotinylated UTP and CTP (Affymetrix) were incorporated in the cRNA, resulting in approximately 1800-fold amplification of labeled RNA.
|
| |
|
| Hybridization protocol |
The target cRNA generated from each sample was fragmented and 15 micogram of the fragmented cRNA was added to the hybridization cocktail which includes in addition to the fragmented target, probe array controls (pre-mixed biotin-labeled bioB, bioC, bioD and cre, in staggered concentrations, ready to be added directly to the hybridization cocktail. These controls facilitate monitoring of the hybridization process for troubleshooting), BSA, and herring sperm DNA. It was then hybridized to the probe array during a 16-hour incubation at 45 deg (C). The array was then washed and stained on the Affymetrix fluidic station (using Streptavidin-Phycoerythrin which binds to the biotinylated UTP and CTP incorporated previously in the hybridized cRNA).
|
| Scan protocol |
The stained array was scanned on the Affymetrix scanner (450).
|
| Data processing |
The GCOS (Gene Operating System) generates the numerical data from the scanned intensities using the MAS 5 software (MicroArray suite 5.0; Affymetrix).
|
| |
|
| Submission date |
Dec 13, 2013 |
| Last update date |
Dec 14, 2013 |
| Contact name |
Shahar Alon |
| Organization name |
Tel Aviv University
|
| Department |
Department of Neurobiology, The George S. Wise Faculty of Life Sciences
|
| Lab |
Yoav Gothilf Lab
|
| Street address |
Tel Aviv University
|
| City |
Tel Aviv |
| ZIP/Postal code |
69978 |
| Country |
Israel |
| |
|
| Platform ID |
GPL1319 |
| Series (1) |
| GSE53288 |
Effect of light on gene expression in the zebrafish pineal gland |
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