|
| Status |
Public on Jul 08, 2011 |
| Title |
Treated_2b |
| Sample type |
RNA |
| |
|
| Source name |
Treated Embryo Sample 2B (Treated Embryo sample 2 Replicate)
|
| Organism |
Danio rerio |
| Characteristics |
agent: 50nM thyroid hormone
development stage: 5 dpf's embryo
|
| Treatment protocol |
Three treated embryo replicates and three control replicates with 100 newwly-hatched larvae (48 hpf) were exposed for 72h in a 100ml glass. Treated solutions were prepared with 50nM of 3.3',5-triiodo-L-throixine (T3) dissoved in DMSO and the control solutions were prepared with the same final concentration of DMSO (0,1%) and directly prepared in embryo water. All the solutions were prepared on the same day of the experiment.
|
| Growth protocol |
Zebrafish embryos were obtained by natural mating and raised at 28.5ºC with 12l:12D photoperiode in embryo water [90mg/l of instant ocean (aquarium systems, sorreburg,France), 0,58 CaSO4·2H2O, dissolved in reverse osmosis purified water].
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from 100 embryos per replicate using Trizol reagent protocol (Invitrogen. Rna concentration was measured by spectrophotometric absorption at 260nm in nanodrop ND-800 (nanodrop technologies, Wilmington, DE) and their quality checked by Agilent 2100 bioanalyzer (agilent technologies, Santa Clara CA).
|
| 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).
|
| |
|
| 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 55 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 Agilent-015064 D. rerio (Zebrafish) Oligo Microarray 4x44K G2519F (Platform GPL7302) 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 (G2505BA) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after 72hr in 50nM T3
T2B-M
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_95 and Grid: 019161_D_F_20080131) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Jul 29, 2010 |
| Last update date |
Jul 09, 2011 |
| Contact name |
Sergi Pelayo Martinez |
| E-mail(s) |
spmbmc@cid.csic.es
|
| Organization name |
CSIC
|
| Department |
IDAEA
|
| Lab |
502
|
| Street address |
Jordi Girona n.18
|
| City |
Barcelona |
| State/province |
Barcelona |
| ZIP/Postal code |
08034 |
| Country |
Spain |
| |
|
| Platform ID |
GPL7301 |
| Series (1) |
| GSE23315 |
Quantitative analysis of thyroid response in zebrafish embryos |
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