|
| Status |
Public on Oct 31, 2015 |
| Title |
In vitro_48hr_30nM EE2_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Primary hepatocytes, 48hr, 30nM EE2, replicate 2
|
| Organism |
Oncorhynchus mykiss |
| Characteristics |
tissue: Hepatocytes
developmental stage: Juvenile
|
| Treatment protocol |
Freshly isolated primary rainbow trout hepatocytes were seeded out in 96-well primaria cell plates (500 000 cells/ml) and incubated in 15 degrees at ambient CO2 for 24hr prior to the EE2 exposure. The hepatocytes were then exposed to 0.03, 0.3, 3 and 30 nM EE2 for 48h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the Qiagen RNeasy Plus mini kit (Qiagen GmbH, Hilden, Germany) following the manufacturer's recommendations with some modifications. The protocol includes lysis of the hepatocyte cells, and an on-column DNase digestion. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.6 ug RNA using the One-Color Low RNA Input Linear Amplification kit (Agilent) trictly 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 in order to fulfill the criterias for amount of incorperated dye in the cRNA sample. One-Color Microarray-Based Gene Expression analysis (Low input Quick Amp Labeling), Version 6.5 May 2010
|
| |
|
| Hybridization protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Scan protocol |
Agilent Feature Extraction software v10.7
|
| Description |
Gene expression after 48hr of in vitro exposed hepatocytes to 30nM EE2
|
| Data processing |
The raw data was subjected to normalization, including correction for background signal, flagged for missing and low quality features followed by statistical analysis using GeneSpring GX v12.6 (Agilent technologies).
|
| |
|
| Submission date |
Apr 28, 2015 |
| Last update date |
Oct 31, 2015 |
| Contact name |
Maria Therese Hultman |
| E-mail(s) |
[email protected]
|
| Organization name |
NIVA
|
| Department |
Ecotoxicology
|
| Lab |
Ecotoxicology
|
| Street address |
Gaustadalléen 21
|
| City |
Oslo |
| ZIP/Postal code |
N-0349 |
| Country |
Norway |
| |
|
| Platform ID |
GPL18864 |
| Series (1) |
| GSE68335 |
Ethynylestradiol (EE2) effect on global gene expression in primary rainbow trout (Oncorhynchus mykiss) hepatocytes |
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