|
| Status |
Public on Dec 15, 2013 |
| Title |
Warm treated salmon 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
heart; adult; warm temperature challenged; exercised
|
| Organism |
Oncorhynchus nerka |
| Characteristics |
tissue: heart
treatment: Warm treated
|
| Treatment protocol |
NA
|
| Growth protocol |
NA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Ambion MagMax-96 for microarray following non-spin method provided by manufacturer's instructions
|
| Label |
Alexa555
|
| Label protocol |
1 µg of total RNA was amplified using Ambion MessageAmp II-96 kit and Amplified RNA (5 ug) were reverse-transcribed into cDNA and labelled with Alexa dyes using a SuperScript Plus Indirect Labeling Kit (Invitrogen) according to manufacturer’s instructions. Experimental samples were fluorescently tagged with Alexa 555 (green) and the reference labelled with Alexa 647 (red).
|
| |
|
| Channel 2 |
| Source name |
heart; pooled reference sample
|
| Organism |
Oncorhynchus nerka |
| Characteristics |
tissue: heart
reference: Pool of cold and warm treated coho salmon
|
| Treatment protocol |
NA
|
| Growth protocol |
NA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Ambion MagMax-96 for microarray following non-spin method provided by manufacturer's instructions
|
| Label |
Alexa647
|
| Label protocol |
1 µg of total RNA was amplified using Ambion MessageAmp II-96 kit and Amplified RNA (5 ug) were reverse-transcribed into cDNA and labelled with Alexa dyes using a SuperScript Plus Indirect Labeling Kit (Invitrogen) according to manufacturer’s instructions. Experimental samples were fluorescently tagged with Alexa 555 (green) and the reference labelled with Alexa 647 (red).
|
| |
|
| |
| Hybridization protocol |
Slide processing steps within the Tecan hybridisation station were conducted as follows: one 30 s slide wash step (0.014% SDS) at 23 °C with a 30 s soak time, two 30 s slide wash steps (sdH2O) at 23 °C with 30 s soak times, 3 min slide denaturation at 85 °C with agitation,1 min wash (5x SSC, 0.01% SDS, 0.2% BSA) at 43 °C with a 30 s soak time, 1 h pre-hybridization at 43 °C with medium agitation, two 1.5 min washes (water) at 43 °C with a 30 s soak time, followed by 1 minwash (8x SSC, 0.1% SDS, 4X Denhardt's) at 43 °C, sample injection at 43 °C with agitation, hybridization for 16 h at 43 °C in high viscosity mode with low frequency agitation, four 30 s wash steps, two at 43 °C (2x SSC, 0.01% SDS) followed by two at 23 °C (0.2x SSC), and slide drying at 23 °C for 2.5 min.
|
| Scan protocol |
A ScanArray Express microarray scanner (Perkin Elmer) was used to capture fluorescent images, adjusting the PMT gain for optimized visualization of each slide.
|
| Description |
biological replicate 1 of 4 (warm)
|
| Data processing |
The images were quantified using Imagene (BioDiscovery, El Segundo, CA, www.biodiscovery.com) and spots with poor quality or no signal (b2 standard deviations from background) at both wavelengths were flagged. Raw microarray intensity data were normalized in GeneSight (version 4.1, BioDiscovery, Inc., www.biodiscovery.com) using the local intensity-dependent loess normalization in order to remove intensity-dependent dye bias (Yang et al., 2002). Flagged spots were replaced by the experimental means for each gene.
All data were log transformed (base 2) and an intensity ratio was computed by taking the differences in log transformed intensities between the sample and reference control. These log-transformed intensity ratios were used in all further analyses. Microarray data were expressed in terms of mean normalized (background corrected) log2 ratios between each fish and the reference control.
|
| |
|
| Submission date |
Aug 21, 2013 |
| Last update date |
Dec 15, 2013 |
| Contact name |
Shaorong Li |
| E-mail(s) |
Shaorong.Li@dfo-mpo.gc.ca
|
| Organization name |
Fisheries and Oceans Canada
|
| Department |
Salmon and Freshwater Ecosystems Division
|
| Lab |
Molecular Genetics Lab
|
| Street address |
3190 Hammond Bay Road
|
| City |
Nanaimo |
| State/province |
British Columbia |
| ZIP/Postal code |
V9T 6N7 |
| Country |
Canada |
| |
|
| Platform ID |
GPL3976 |
| Series (1) |
| GSE50054 |
Facing warm temperatures during migration – cardiac mRNA responses of two adult sockeye salmon Oncorhynchus nerka populations to warming and swimming challenges |
|
