|
| Status |
Public on Aug 25, 2017 |
| Title |
[6] Photo-Ox IMX-101, 58.2, Rep 1 |
| Sample type |
RNA |
| |
|
| Source name |
whole animal
|
| Organism |
Danio rerio |
| Characteristics |
tissue: larvae
concentration: 58.2
treatment: Photo-Ox IMX-101
|
| Treatment protocol |
Zebrafish larvae were exposed to IMX-101 at 0, 3.8, 30.2 and 58.2 mg/L (measured concentrations) as the parent or UV-irradiated IMX-101. Danio rerio larvae were exposed in static nonrenewal acute 96-h bioassays. Exposure chambers were 300-ml high-form lipless beakers with a test solution volume of 250 ml. Ten larval fish per beaker were exposed in the parent or UV-treated IMX-101 where all treatments included 4 exposure replicates. Larval zebrafish were fed newly hatched Artemia nauplii two hours before the initiation of exposures and at the 48-h timepoint. Surviving fish were enumerated daily and any fish found to be deceased were promptly removed from exposure chambers.
|
| Growth protocol |
Exposure chambers were 300-ml high-form lipless beakers with a test solution volume of 250 ml. Ten larval fish per beaker were exposed in the parent and UV-treated IMX-101 exposures where all treatments included 4 exposure replicates. Larval zebrafish were fed newly hatched Artemia nauplii two hours before the initiation of exposures and at the 48-h timepoint.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from 4 whole fish/replicate for all transcript expression experiments. All samples were flash frozen upon collection and stored at -80 °C until RNA extraction. Samples were homogenized in lysis buffer with a FAST Prep-24 instrument (MP Biomedicals, Santa Ana, CA) before RNA isolation with RNeasy kits (Qiagen, Valencia, CA). Total RNA concentrations were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop technologies, Wilmington, DE, USA). The integrity of total RNA was assessed on an Agilent 2100 Bioanalyzer (Palo Alto, CA). Criteria for acceptable RNA integrity included a RNA integrity number >7.0 from the Agilent 2100 Bioanalyzer and a 260/280 spectrophotometric reading >2.0.
|
| Label |
Cy3
|
| Label protocol |
The Agilent Low-Input Quick Amp, One Color Microarray Hybridization protocol (Agilent Technologies) was utilized following manufacturer’s recommendations where 600 ng of total RNA served as the starting material for each biological replicate.
|
| |
|
| Hybridization protocol |
All samples were hybridized to Agilent 8 x 60K, zebrafish microarrays (AMADID #36575). Microarray hybridization experimental designs for IMX-101 exposures were completely randomized (using a random number generator) with factorial treatment arrangements contrasting parent versus UV-treated chemicals across each concentration series. Exposure concentrations were selected to represent both sublethal concentrations and concentrations beyond the threshold of lethality to facilitate identification of mechanisms of toxicity. The IMX-101 experiment included UV-treated and parent IMX-101 at the measured exposure concentrations: 3.8, 30.2 and 58.2 mg/L (measured concentrations) each including 4 exposure replicates, in addition to 4 control (0 mg/L) exposure replicates. Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to the custom-designed Agilent test array for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed at room temperature with GE Wash Buffer 1 (Agilent) and with 37°C GE Wash buffer 2 (Agilent).
|
| Scan protocol |
An Agilent Surescan Microarray Scanner (G2505 C, Agilent Technologies Inc.) was used to scan microarrays at 2 μm resolution.
|
| Data processing |
Data were extracted from microarray images using Agilent Feature Extraction software (Agilent Technologies). Analysis of internal control spikes indicated that signal data was within the linear range of detection. Microarray data were normalized to the 75th percentile within each array followed by median scaling among all exposures using GeneSpring Software version GX14.5 (Agilent Technologies). GeneSpring was additionally used to conduct ANOVA-based differential transcript expression analyses. Two-way ANOVA was utilized to test the interactive potential between the UV-treatment and IMX-101 exposures on transcript expression. In the two-way ANOVA tests a p-value cutoff of 0.005 was utilized to identify significantly affected transcripts for the IMX-101 exposure, the UV treatment and the IMX-101 x UV treatment interaction. In order to identify the specific differences in transcript expression between the parent IMX-101 and the UV-treated IMX-101, post hoc pairwise tests were conducted for all significant targets identified in the UV treatment and the IMX-101 x UV treatment interaction. The post hoc analysis tested the effects of the UV-treated IMX-101 relative to the parent IMX-101 at each dose-level using a moderated t-test (p = 0.05) and a 1.5 fold change cut-off.
|
| |
|
| Submission date |
Feb 07, 2017 |
| Last update date |
Aug 25, 2017 |
| Contact name |
Kurt A Gust |
| E-mail(s) |
[email protected]
|
| Phone |
601-634-3593
|
| Organization name |
US Army ERDC
|
| Department |
Environmental Laboratory
|
| Lab |
Environmental Genomics and Systems Biology Team
|
| Street address |
3909 Halls Ferry Rd.
|
| City |
Vicksburg |
| State/province |
MS |
| ZIP/Postal code |
39180 |
| Country |
USA |
| |
|
| Platform ID |
GPL23036 |
| Series (2) |
| GSE94621 |
The Increased Toxicity of UV-Degraded Nitroguanidine and IMX-101 Exposures in Zebrafish Larvae: Evidence Implicating Oxidative Stress [6] |
| GSE94700 |
The Increased Toxicity of UV-Degraded Nitroguanidine and IMX-101 Exposures in Zebrafish Larvae: Evidence Implicating Oxidative Stress |
|
