|
| Status |
Public on Feb 02, 2009 |
| Title |
Sample 81 - Testis exposed to 3 mg O2/L for 4 d |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Sample 81 - Testis exposed to 3 mg O2/L for 4 d
|
| Organism |
Danio rerio |
| Characteristics |
Gender: Male
Age: 7 months
Strain: ab wild-type strain
Tissue: Gonad
Sample: Total RNA
|
| Biomaterial provider |
from on-site culture unit at US EPA, MED – Duluth, MN, USA
|
| Treatment protocol |
fish were humanely euthanized in buffered tricaine methanesulfonate (Finquel, Argent, Redmond, WA, USA).
|
| Growth protocol |
25°C, 16:8 light:dark photoperiod, fed live Artemia
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Gonads were thawed, homogenized and total RNA extracted with TRI Reagent® (Sigma-Aldrich, St. Louis, MO) following the manufacturer’s protocol. Total RNA was quantified using a nanodrop ND-100 spectrophotometer (Nanodrop technologies, Wilmington, DE). Integrity and purity of RNA samples was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Paulo Alto, CA) and an RNA Pico Chip Kit (Agilent).
|
| Label |
Cy5
|
| Label protocol |
RNA from each condition of interest (treatment) was labeled with a Cy5 dye and hybridized onto arrays together with Cy3 labeled RNA taken from a standard reference pool of RNA extracted from sexually mature male and female gonads and brains, and female livers. For the microarray analyses, total RNA was amplified using Agilent low input linear amplification kit according to the process outlined by the manufacturer (Agilent Technologies). Amplified target cRNA (1-5 µg) was labeled with either cyanine-5 or cyanine-3 using ULS aRNA Flurorescent Labeling Kits from Kreatech according to the manufacturers protocol (Kreatech Biotechnology, Amsterdam, The Netherlands). Concentrations of labeled cRNA and the label incorporation was determined using Nanodrop-1000 spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
Reference - pool of gonads and brains from males and females, and livers from females
|
| Organism |
Danio rerio |
| Characteristics |
Gender: Females and Males
Age: 7 months
Strain: ab wild-type strain
Tissue: gonads and brains from males and females, livers from females
Sample: Total RNA
|
| Biomaterial provider |
from on-site culture unit at US EPA, MED – Duluth, MN, USA
|
| Treatment protocol |
fish were humanely euthanized in buffered tricaine methanesulfonate (Finquel, Argent, Redmond, WA, USA).
|
| Growth protocol |
25°C, 16:8 light:dark photoperiod, fed live Artemia
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissues were thawed, homogenized and total RNA extracted with TRI Reagent® (Sigma-Aldrich, St. Louis, MO) following the manufacturer’s protocol. Total RNA was quantified using a nanodrop ND-100 spectrophotometer (Nanodrop technologies, Wilmington, DE). Integrity and purity of RNA samples was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Paulo Alto, CA) and an RNA Pico Chip Kit (Agilent).
|
| Label |
Cy3
|
| Label protocol |
RNA from each condition of interest (treatment) was labeled with a Cy5 dye and hybridized onto arrays together with Cy3 labeled RNA taken from a standard reference pool of RNA extracted from sexually mature male and female gonads and brains, and female livers. For the microarray analyses, total RNA was amplified using Agilent low input linear amplification kit according to the process outlined by the manufacturer (Agilent Technologies). Amplified target cRNA (1-5 µg) was labeled with either cyanine-5 or cyanine-3 using ULS aRNA Flurorescent Labeling Kits from Kreatech according to the manufacturers protocol (Kreatech Biotechnology, Amsterdam, The Netherlands). Concentrations of labeled cRNA and the label incorporation was determined using Nanodrop-1000 spectrophotometer
|
| |
|
| |
| Hybridization protocol |
All of the labeling and post labeling procedures were conducted in ozone-free enclosure to ensure the integrity of the label. Labeled materials were set up for the fragmentation reaction according to Agilent protocol described in their processing manual, and hybridized overnight in the rotating oven at 65°C in an ozone-free room. The Agilent Oligo Microarray Zebrafish slide, with approximately 21, 000 genes represented was used for the gene expression analyses (4 x 44K format, product 015064). Labeled samples (825 ng) were co-hybridized using sureHyb chambers from Agilent. Wash conditions used were as outlined in the Agilent processing manual (protocol version 4.0).
|
| Scan protocol |
The arrays were scanned using Agilent scanner (G2505B). Agilent feature extraction software was used for extracting array data; the initial data quality analyses were done using Rosetta Luminator software (Rosetta Biosoftware, Seattle, WA).
|
| Description |
Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
|
| Data processing |
To generate lists of differentially expressed genes, output from Agilent’s feature extraction software was imported using GeneSpring GX 7.3.1 software (Agilent Technologies). The enhanced Agilent FE Import function was used to flag low quality spots based on feature saturation, uniformity, pixel population consistency and signal-to-noise ratio using procedures described in the Agilent Technologies Training Manual (version 4.5, May 2006). The ratios of treatment and reference raw intensity data then were log transformed and normalized using locally weighted least squares regression (lowess) normalization, available in GeneSpring GX 7.3.1.
|
| |
|
| Submission date |
Mar 20, 2008 |
| Last update date |
Feb 02, 2009 |
| Contact name |
Dalma Martinovic |
| E-mail(s) |
Martinovic.Dalma@epa.gov
|
| Organization name |
U.S. EPA
|
| Department |
ORD-NHEERL
|
| Lab |
MED
|
| Street address |
6201 Congdon Blvd
|
| City |
Duluth |
| State/province |
MN |
| ZIP/Postal code |
55812 |
| Country |
USA |
| |
|
| Platform ID |
GPL6563 |
| Series (1) |
| GSE10951 |
Hypoxia alters gene expression in the gonads of zebrafish |
|
