|
| Status |
Public on Apr 21, 2016 |
| Title |
Larvae_3 dpf_3 ppb_replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
Larvae_3 ppb
|
| Organism |
Danio rerio |
| Characteristics |
tissue: Larvae
age: 3 dpf
treated with: 3 parts per billion (ppb) atrazine
|
| Treatment protocol |
Embryos were dosed with 0, 0.3, 3, or 30 ppb atrazine (CAS #1912-24-9; Chem Service, 98% purity) from 1-72 hours post fertilization (hpf) as previously described (Weber et al. 2013; Wirbisky et al., 2015). After the exposure, total RNA was extracted and miRNA microarray was peformed.
|
| 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 470-520 µS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA (whole zebrafish larvae) was isolated by the miRNeasy Kit (Qiagen, Venlo, Limburg).
|
| Label |
Cy3
|
| Label protocol |
Samples were dephosphorylated using Calf Intestinal Phosphatase and then denatured with dimethyl sulfoxide. Following denaturation, samples were cooled and subsequently labeled with Cyanine 3-pCp using a T4 RNA Ligase. Labeled samples were purified using MicroBioSpin 6 columns (Bio-Rad, Hercules, CA) and dried using a Savant Speed Vac on medium-high heat for 1 hour.
|
| |
|
| Hybridization protocol |
Dried samples were resuspended in nuclease-free water and prepared for hybridization using GE Blocking Agent and Hi-RPM Hybridization Buffer. Samples were loaded onto a custom designed 8X60K Agilent miRNA array in which all human and zebrafish miRNAs were included (based on miRBase release 18.0). Arrays were loaded into SureHyb chambers and hybridized at 55oC for 20 hours in an Agilent Microarray Hybridization Oven.
|
| Scan protocol |
The following day, arrays were removed from the oven, washed with Agilent Gene Expression Wash Buffers and scanned on an Agilent SureScan Microarray scanner.
|
| Description |
L_R2_3
|
| Data processing |
Probe information was extracted from generated tif images using Feature Extraction image analysis software 9.5.3 using QC Metric Set to evaluate the quality of labeling and hybridization. Data were background-subtracted, normalized to the 90th percentile, and analyzed for differential expression in GeneSpring 12.5 software using a one-way analysis of variance (ANOVA) with a Tukey’s post hoc test (p<0.05) to determine which atrazine treatment groups were different from the control.
|
| |
|
| Submission date |
Mar 01, 2016 |
| 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 |
GPL21545 |
| Series (1) |
| GSE78805 |
Embryonic atrazine exposure alters zebrafish and human miRNAs associated with angiogenesis, cancer, and neurodevelopment |
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