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| Status |
Public on Jul 23, 2011 |
| Title |
brain_14d_273 ug/L diazepam_replicate 4 |
| Sample type |
RNA |
| |
|
| Source name |
Male zebrafish, 14 d, 273 ug/L diazepam, replicate 4
|
| Organism |
Danio rerio |
| Characteristics |
tissue: brain
developmental stage: male
age: adult
treatment: 273 ug/L diazepam
|
| Treatment protocol |
The experimental setup consisted of four replicates of water controls and two diazepam doses. Twenty male zebrafish per replicate were held in 20-L stainless steel tanks in well-aerated exposure water. Fish were exposed for 14 d to the nominal concentrations of 273 ng/L and 273 μg/L diazepam, respectively. The lower concentration (273 ng/L) was assumed to represent the worst case environmental concentration, and 273 μg/L refers to the NOEC in the early life stages test (12). During the experiment fish survival, appearance and behavior were determined. A 48 h static-renewal procedure was used to minimize handling stress for the fish. Thereby after 24 h, food remains and feces were removed by siphoning 1/3 of the water and replacement by new exposure water containing the appropriate diazepam concentrations. After 48 h, the water renewal procedure was repeated, but this time by replacing total tank water (20 L). The quality of the exposure water was continuously monitored by the oxygen concentration determination (>70%), the pH value (6.7-7.2) and the temperature (27±1 °C).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from male zebrafish brains using the RNeasy Mini Kit (Qiagen, Basel, Switzerland). Total RNA concentrations were measured spectrophotometrically using a NanoDrop ND-1000 UV-VIS Spectrophotometer at 260 nm. The integrity of each RNA sample was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Basel, Switzerland). Only samples containing a 260/280 nm ratio between 1.8-2.1, a 28S/18S ratio between 1.5-2 and an RNA integrity number (RIN) > 8 were processed further.
|
| Label |
Cy3
|
| Label protocol |
Total RNA samples (600 ng) were reverse-transcribed into double-strand cDNA in the presence of RNA poly-A controls with the Agilent One-Color RNA Spike-In Kit. Cy3 labeling and hybridization were performed according to the manufacturer's manual. After reverse-transcription of RNA into double-stranded cDNA, double-strand cDNA was in vitro transcribed into cRNA in the presence of Cy3 labeled nucleotides using a Low RNA Input Linear Amp Kit +Cy dye (Agilent Technologies, Basel, Switzerland). The Cy3-labeled cRNA was purified using an RNeasy mini kit (Qiagen, Basel, Switzerland), and quality and quantity was determined using a NanoDrop ND-1000 UV-VIS Spectrophotometer and an Agilent 2100 Bioanalyzer, respectively. Only cRNA samples with a total cRNA yield higher than 2 µg and a dye incorporation rate between 9 pmol/µg and 20 pmol/µg were used for hybridization.
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| |
|
| Hybridization protocol |
Cy-3-labeled cRNA samples (1.65 µg) were mixed with Agilent blocking solution, subsequently fragmented randomly to 100-200 bp at 65 °C with fragmentation buffer and resuspended in hybridization buffer as provided by the gene expression hybridization Kit (Agilent Technologies). Target cRNA samples (100 µL) were hybridized to the Agilent Zebrafish 4x44K Gene Expression Microarray for 17 h at 65 °C. The hybridized arrays were then washed using Agilent GE wash buffers 1 and 2 according to the manufacturer's instructions.
|
| Scan protocol |
Slides were scanned by an Agilent Microarray Scanner (Agilent p/n G2565BA) at 5 μm resolution with the green photomultiplier tube set to 100% and a scan area of 61 x 21.6 mm.
|
| Description |
Gene expression after 14 d exposure to 273 ug/L diazepam
|
| Data processing |
Image generation and feature extraction was performed using the Agilent Feature Extraction (FE) software version 9.5.3. Quality control was additionally considered before performing the statistical analysis. These included array hybridization pattern inspection: absence of scratches, bubbles, areas of non-hybridization, proper grid alignment, spike performance in controls with a linear dynamic range of 5 orders of magnitude and the number of green-feature non-uniformity outliers which should be below 100 for all samples.
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| |
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| Submission date |
Jul 26, 2010 |
| Last update date |
Jul 23, 2011 |
| Contact name |
Daniela Maria Oggier |
| Organization name |
University of Applied Sciences Northwestern Switzerland
|
| Department |
School of Lifesciences
|
| Street address |
Gründenstrasse 40
|
| City |
Muttenz |
| ZIP/Postal code |
4132 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL6457 |
| Series (1) |
| GSE23157 |
Effects of diazepam on gene expression and link to physiological effects in different life stages in zebrafish Danio rerio |
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