|
| Status |
Public on Nov 12, 2013 |
| Title |
Zebrafish_self_to_self_rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
pooled embryos control G3
|
| Organism |
Danio rerio |
| Characteristics |
developmental stage: 2 dpf embryo
agent: water control
|
| Treatment protocol |
Three treated embryo replicates and three control replicates with 50 fertilized eggs from the 4-cell stage to the 128-cell stage were exposed for 48h in a 100ml glass. Treated solutions were prepared with 0.05uM of PAMAM dendrimer Generation 3 dissolved in water for fish and the control solutions treatments were prepared and directly prepared in embryo water. All the solutions were prepared on the same day of the experiment and renewal each day. Three treated embryo replicates and three control replicates with 50 fertilized eggs from the 4-cell stage to the 128-cell stage were exposed for 48h in a 100ml glass. Treated solutions were prepared with 0.5uM of PAMAM dendrimer Generation 4 dissolved in water for fish and the control solutions treatments were prepared and directly prepared in embryo water. All the solutions were prepared on the same day of the experiment and renewal each day.
|
| Growth protocol |
Zebrafish (D. rerio) embryos and eleutheroembryos were obtained by natural mating and raised at 28.5 ◦C (Kimmel et al., 1995) with a 12L:12D photoperiod in embryo water [90 g/mL of Instant Ocean (Aquarium Systems, Sarrebourg, France; http://zfin.org/), 0.58 mM CaSO4·2H2O, dissolved in reverse osmosis purified water]. Animal stages were recorded as days or hours postfertilization (dpf or hpf).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from 50 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 integrity checked by Agilent 2100 bioanalyzer (agilent technologies, Santa Clara CA).
|
| Label |
Cy5
|
| Label protocol |
Cyanine 3-CTP samples and Cyanine 5-CTP samples were prepare from 200ng of total RNA using the Two-Color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labeling kit (Agilent), followed by the purification of the labelled/amplified RNA using Qiagen RNeasy mini kit.
|
| |
|
| Channel 2 |
| Source name |
pooled embryos control G3
|
| Organism |
Danio rerio |
| Characteristics |
developmental stage: 2 dpf embryo
agent: Water control
|
| Treatment protocol |
Three treated embryo replicates and three control replicates with 50 fertilized eggs from the 4-cell stage to the 128-cell stage were exposed for 48h in a 100ml glass. Treated solutions were prepared with 0.05uM of PAMAM dendrimer Generation 3 dissolved in water for fish and the control solutions treatments were prepared and directly prepared in embryo water. All the solutions were prepared on the same day of the experiment and renewal each day. Three treated embryo replicates and three control replicates with 50 fertilized eggs from the 4-cell stage to the 128-cell stage were exposed for 48h in a 100ml glass. Treated solutions were prepared with 0.5uM of PAMAM dendrimer Generation 4 dissolved in water for fish and the control solutions treatments were prepared and directly prepared in embryo water. All the solutions were prepared on the same day of the experiment and renewal each day.
|
| Growth protocol |
Zebrafish (D. rerio) embryos and eleutheroembryos were obtained by natural mating and raised at 28.5 ◦C (Kimmel et al., 1995) with a 12L:12D photoperiod in embryo water [90 g/mL of Instant Ocean (Aquarium Systems, Sarrebourg, France; http://zfin.org/), 0.58 mM CaSO4·2H2O, dissolved in reverse osmosis purified water]. Animal stages were recorded as days or hours postfertilization (dpf or hpf).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from 50 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 integrity checked by Agilent 2100 bioanalyzer (agilent technologies, Santa Clara CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine 3-CTP samples and Cyanine 5-CTP samples were prepare from 200ng of total RNA using the Two-Color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labeling kit (Agilent), followed by the purification of the labelled/amplified RNA using Qiagen RNeasy mini kit.
|
| |
|
| |
| Hybridization protocol |
These following components were added in this order: 825ng of Cy3 labeled cRNA, 825 of Cy5 labeled cRNA, 11ul of 10x blocking Reagent, a volume of nuclease free water up to 52.8ul and 2.2ul of 25x fragmentation buffer. Incubate at 60ºC for exactly 30 minutes. Immediately cool on ice. Stop reaction by adding 55ul of 2x GEx Hybridization Buffer. 100ml of hybridization cocktail was used to each array. Set the Agilent hybridization oven at 65ºC and rotation speed to 10rpm, during 17hours. After hybridization, slides were washed. One last Acetonitrile wash was perform, followed by a stabilization and dry solution wash.
|
| Scan protocol |
Microarrays were scanned using an Agilent DNA Microarray Scanner G2505C, and data were acquired using Agilent's Feature Extraction Software version 10.5.1.1
|
| Description |
G3 control embryo vs G3 control embryo at 2 dpf. Self to self 3
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software v 10.5.1.1 (Agilent) using default parameters (protocol GE2_105_jan09 and Grid: 026437_D_F_20110614) to obtain background subtracted and spatially detrended Processed Signal intensities
|
| |
|
| Submission date |
Oct 10, 2012 |
| Last update date |
Nov 13, 2013 |
| Contact name |
Eva Lima Oliveira |
| E-mail(s) |
olieva@gmail.com
|
| Phone |
003493 400 6100
|
| Organization name |
IDAEA CSIC
|
| Department |
Environmental Chemistry
|
| Lab |
Environmental Toxicology
|
| Street address |
Carrer Jordi Girona 18-26
|
| City |
Barcelona |
| State/province |
Barcelona |
| ZIP/Postal code |
08034 |
| Country |
Spain |
| |
|
| Platform ID |
GPL14688 |
| Series (1) |
| GSE41333 |
Transcriptomic response of zebrafish embryos to Polyamidoamine (PAMAM) dendrimers |
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