|
| Status |
Public on Dec 14, 2010 |
| Title |
larva_30d_mixed_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
larva, 30d, mixed sex, replicate 2
|
| Organism |
Danio rerio |
| Characteristics |
strain: wild type
developmental stage: larva
developmental timing: 30d
gender: mixed
number of individuals per sample: 5
|
| Treatment protocol |
Staging was done according to post-fertilization time. Embryos were additionally staged under the dissecting microscope according to ref. 1 to check for the consistency of post-fertilization timing and morphological development at standard temperature (28.5 °C). Only healthy animals that showed the expected morphological features for a given post-fertilization time were sampled. Each sample contained around 50 individuals until the 1 day and 3 hour embryo stage, 15 individuals until the 10 day larval stage, 10 individuals until the 18 day larval stage, 5 individuals until the 45 day juvenile stage, while in later juvenile and adult stages we sampled males and females separately and each sample contained 2 individuals. All samples were snap frozen in liquid nitrogen and stored at -80 °C until RNA extraction. To avoid severe biases due to the excess of unfertilized eggs, we squeezed eggs from adult females before freezing them in liquid nitrogen. 1. (Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development of the zebrafish. Dev Dyn. 1995 Jul;203(3):253-310.)
|
| Growth protocol |
Zebrafish (Danio rerio) were kept in 12L flow-through tanks at 26.5 °C (around 60 animals per tank). For accurate staging, fertilized eggs were collected within 15 min intervals and incubated in Petri dishes at 28.5°C with water changes every 2-6 hours. After hatching, larvae were transferred to 1L tanks and kept at 28.5 °C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen tissues were homogenized in TRIZOL (Invitrogen) by shaking samples 4 min together with a 5mm steel bead in a bead mill (TissueLyser II, Qiagen) at 30 Hz (embryos and smaller larve) or by disruption (larger larvae, juveniles and adults) using rotor-stator homogenizer (Tissue Ruptor, Qiagen). Total RNA was isolated using TRIZOL plus protocol from Invitrogen and the quality and the yield was determined by BioAnalyzer 2100 (Agilent) and NanoDrop ND-1000 spectrophotometer (Thermo scientific)
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 400 ng RNA using the Agilent one-color Quick Amp Labeling Kit protocol 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.
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| |
|
| Hybridization protocol |
1650 ng of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Zebrafish (V2) Gene Expression Microarray slides (4x44K) (G2519F) 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 34°C GE Wash buffer 2 (Agilent), then dried 1 minute at room temperature.
|
| Scan protocol |
Slides were scanned immediately after washing and drying on the Agilent High-Resolution Microarray Scanner (G2505C) using one color scan setting for 4x44k array slides (Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
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| Description |
Agilent Feature Extraction file:
US84303580_251916110554_S01_GE1_105_Dec08_1_3.txt.
Gene expression in wild type zebrafish larva, 30d, mixed sex, biological replicate 2.
|
| Data processing |
The scanned images were analyzed with Feature Extraction 10.7 Image Analysis Software (Agilent) using default parameters (protocol GE1_105_Dec08 and Grid: 019161_D_F_20080627) to obtain background subtracted and spatially detrended Processed Signal intensities.
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| |
|
| Submission date |
Oct 11, 2010 |
| Last update date |
Dec 14, 2010 |
| Contact name |
Tomislav Domazet-Loso |
| E-mail(s) |
tdomazet@irb.hr
|
| Organization name |
Ruder Boskovic Institute
|
| Department |
Division of Molecluar Biology
|
| Lab |
Laboratory of Evolutionary Genetics
|
| Street address |
Bijenicka cesta 54
|
| City |
Zagreb |
| ZIP/Postal code |
10000 |
| Country |
Croatia |
| |
|
| Platform ID |
GPL6457 |
| Series (1) |
| GSE24616 |
A phylogenetically based transcriptome age index mirrors ontogenetic divergence patterns |
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