|
| Status |
Public on Dec 31, 2012 |
| Title |
CMO-Male-Liver-Replicate2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total liver RNA from CMO exposed male FHM labeled with Cyanine-5 (red)
|
| Organism |
Pimephales promelas |
| Characteristics |
gender: male
developmental stage: sexually mature adult
tissue: liver
treatment: 100% final effluent
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation was performed using the manufacturer’s recommended ratio of 1 ml TRIzol reagent (Invitrogen) per 100 mg of liver.
|
| Label |
Cy5
|
| Label protocol |
The RNA samples were labeled using Agilent’s Quick-AMP 2-Color Labeling kit (#5190-0444). Each experimental array sample was prepared by combining 825 ng of Cy5 labelled liver RNA sample with 825 ng of the Cy3 labelled reference sample.
|
| |
|
| Channel 2 |
| Source name |
Pooled reference sample from all river water exposed (control) FHM (3 female and 3 male)
|
| Organism |
Pimephales promelas |
| Characteristics |
developmental stage: sexually mature adult
tissue: liver
treatment: reference pool
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation was performed using the manufacturer’s recommended ratio of 1 ml TRIzol reagent (Invitrogen) per 100 mg of liver.
|
| Label |
Cy3
|
| Label protocol |
The RNA samples were labeled using Agilent’s Quick-AMP 2-Color Labeling kit (#5190-0444). Each experimental array sample was prepared by combining 825 ng of Cy5 labelled liver RNA sample with 825 ng of the Cy3 labelled reference sample.
|
| |
|
| |
| Hybridization protocol |
Agilent manufactured Fathead minnow microarrays based on the EcoArray platform (22K unique probes, GLP7282) were hybridized in Agilent hybridization chambers and incubated overnight at 65°C. The slides were washed for 1 min in 6X SSPE, 0.005% N-Laurosylsarcosine; 1 min in 0.06X SSPE, 0.005% N-Laurosylsarcosine; and then dried by dipping the slides in two trays containing a stabilization and drying solution for 60 s and 30 s.
|
| Scan protocol |
Slides were scanned with an Agilent DNA microarray scanner, and raw images were processed using Agilent’s Feature Extraction Software, Version 9.5.3.
|
| Data processing |
log2 transformed signal ratio between the experimental channel and the reference channel was calculated for each spot following background correction of the spot intensities. Data for male and female fish were analyzed independently. The raw data were LOWESS normalized within arrays and scaled between arrays using the limma package implemented in the R environment for statistical computing (http://www.R-project.org)
|
| |
|
| Submission date |
Dec 08, 2010 |
| Last update date |
Dec 31, 2012 |
| Contact name |
Shannon L Costigan |
| E-mail(s) |
[email protected]
|
| Organization name |
Lakehead University
|
| Department |
Biology
|
| Lab |
Law
|
| Street address |
955 Oliver Rd
|
| City |
Thunder Bay |
| State/province |
ON |
| ZIP/Postal code |
P7B5E1 |
| Country |
Canada |
| |
|
| Platform ID |
GPL7282 |
| Series (1) |
| GSE25928 |
Gene expression profiling of fathead minnows (Pimephales promelas) following an acute exposure to pulp and paper mill effluents |
|
