|
| Status |
Public on Dec 31, 2019 |
| Title |
9 mpf_brain_male_0ppb_Rep3 |
| Sample type |
RNA |
| |
|
| Source name |
9 mpf, brain, male, 0 ppb, replicate 3
|
| Organism |
Danio rerio |
| Characteristics |
gender: Male
tissue: Brain
age: 9 mpf
atz exposue: 0ppb
|
| Treatment protocol |
Zebrafish embryos were exposed to 0, 0.3, 3, or 30 ppb atrazine from approximately 1 hpf through 72 hpf. Zebrafish were rinsed at 72 hpf and reared in normal conditions until 9 mpf. After euthanasia, brains were removed, homogenized in Trizol (Life Technolgies, Carlsbad, CA) and flash frozen in liquid nitrogen and stored at -80°C until RNA extraction. Each replicate represents one zebrafish brain and four biological replicates were completed (n=4).
|
| Growth protocol |
Zebrafish (wild-type AB strain) were housed in a Z-Mod System (Aquatic Habitats, Apopka, FL) on a 14:10 hour light:dark cycle and maintained at 28ºC ± 1°C with a pH of 7.0-7.2 and conductivity range of 470-550 µS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using an RNeasy MinElute Cleanup Kit (Qiagen Sciences, Maryland, USA) following established protocols. RNA concentrations were measured by using a NanoDrop 1000 spectrophotometer and the quality was verified by absorbance ratio 260/280 nm.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared using the One-Color Low Input QuickAmp Labeling kit (Agilent) 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.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55mL containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturer’s instructions. On completion of the fragmentation reaction, 55 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent custom zebrafish 4X180K Microarrays (060509) 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).
|
| Scan protocol |
Arrays were scanned on an Agilent Technologies SureScan Microarray Scanner (Agilent Technologies, Santa Clara, CA).
|
| Description |
Gene expression 9 mpf following embryonic exposure (1-72 hpf)
|
| Data processing |
Array image data was extracted using Agilent Feature Extraction Software 12.0 (Agilent Technologies, Santa Clara, CA). Array image data was then uploaded to GeneSpring 14.9 (Agilent Technologies, Santa Clara, CA) for statistical analysis. Each gene list was imported into Ingenuity Pathway Analysis (IPA) for gene ontology and molecular pathway analysis.
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| |
|
| Submission date |
Mar 29, 2018 |
| Last update date |
Aug 11, 2022 |
| Contact name |
Jennifer Freeman |
| E-mail(s) |
jfreema@purdue.edu
|
| Organization name |
Purdue University
|
| Department |
School of Health Sciences
|
| Street address |
550 Stadium Mall Drive
|
| City |
West Lafayette |
| State/province |
IN |
| ZIP/Postal code |
47907 |
| Country |
USA |
| |
|
| Platform ID |
GPL23074 |
| Series (2) |
| GSE112504 |
Embryonic Atrazine Exposure Causes Behavioral, Transcriptomic, Epigenetic, and Pathological Alterations in Adult Zebrafish Brain [male] |
| GSE112505 |
Embryonic Atrazine Exposure Causes Behavioral, Transcriptomic, Epigenetic, and Pathological Alterations in Adult Zebrafish Brain |
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