|
| Status |
Public on Nov 01, 2013 |
| Title |
Summer run_Sockeye_19°C_M_Array2_10380 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Gill tissue warm treatment
|
| Organism |
Oncorhynchus nerka |
| Characteristics |
year: 2007
temperature (c): 19
run-timing group: Summer-run
stock: Mitchell
gender: male
tissue: gill
|
| Treatment protocol |
Summer-run sockeye salmon from the Fraser River, B.C., Canada, were exposed to either a warm (19C) or cool (14C) temperature treatment for 7 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Freshly sampled gill tissues were quickly frozen in liquid nitrogen and stored at -80 degrees C until RNA extractions were performed. At each of the subsequent steps leading up to the hybridization of microarray slides, samples were randomized to minimize the impact of technical artefacts. The -80 degrees C samples were removed and processed immediately by adding 600 ml of TRI-reagent and one stainless steel 3 mm ball and homogenized using a Retsch MM301 mixer mill. RNA was extracted using a Biomek FXP automated liquid-handling instrument with the Ambion MagMax-96 for Microarrays Total RNA Isolation Kit, according to the manufacturer's instructions and robotic scripts. Total RNA purity and quantity was assessed by measuring the A260/A280 ratio. Total RNA (11 microlitres) was used to make aaRNA using Ambion Amino Allyl MessageAmpTM II-96 kits. The aaRNA was made using two 96-well plates.
|
| Label |
Alexa 555
|
| Label protocol |
Total RNA (11 microlitres) was used to make aaRNA and labelled with Alexa dyes using the Invitrogen Indirect Labelling Kit. The aaRNA samples were purified, lyophilized and eluted in 12 μl of DNAse/RNAse-free water prior to the dye coupling reactions. During dye labelling, samples were processed individually: DMSO was added to the Alexa dye tube, followed by aaDNA and incubated for 1h at room temperature. After a 1 h incubation at room temperature, the reactions were quenched using 4M hydroxylamine and the samples were purified, eluted in 35 μl of aaRNA elution buffer and quantified using a NanoDrop Spectrophotometer ND-1000. A standard reference design was used in the microarray experiments where the reference was comprised of a mixture of RNA from all individuals used in the study. All individual samples to be run on the arrays were fluorescently tagged with Alexa 555 and the mixed reference sample was labelled with Alexa 647. Prior to hybridization, dye labelled treatment and reference samples (825 ng each) were combined along with 1.2 μl 25X fragmentation buffer, 6 μl blocking agent and nuclease-free water to bring the final volume to 30 μl. The fragmentation mix was incubated at 60°C for 30 minutes and was stopped by adding 30 μl of 2X GEx hybridization buffer HI-RPM. Samples were briefly centrifuged, placed on ice and 55 μl of the mix was loaded into hybridization chambers in a Tecan-HS4800 Pro Hybridization Station.
|
| |
|
| Channel 2 |
| Source name |
a pool of RNA from all of the fish used in the experiment
|
| Organisms |
Oncorhynchus gorbuscha; Oncorhynchus nerka |
| Characteristics |
tissue: gill
sample type: Reference
|
| Treatment protocol |
Summer-run sockeye salmon from the Fraser River, B.C., Canada, were exposed to either a warm (19C) or cool (14C) temperature treatment for 7 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Freshly sampled gill tissues were quickly frozen in liquid nitrogen and stored at -80 degrees C until RNA extractions were performed. At each of the subsequent steps leading up to the hybridization of microarray slides, samples were randomized to minimize the impact of technical artefacts. The -80 degrees C samples were removed and processed immediately by adding 600 ml of TRI-reagent and one stainless steel 3 mm ball and homogenized using a Retsch MM301 mixer mill. RNA was extracted using a Biomek FXP automated liquid-handling instrument with the Ambion MagMax-96 for Microarrays Total RNA Isolation Kit, according to the manufacturer's instructions and robotic scripts. Total RNA purity and quantity was assessed by measuring the A260/A280 ratio. Total RNA (11 microlitres) was used to make aaRNA using Ambion Amino Allyl MessageAmpTM II-96 kits. The aaRNA was made using two 96-well plates.
|
| Label |
Alexa 647
|
| Label protocol |
Total RNA (11 microlitres) was used to make aaRNA and labelled with Alexa dyes using the Invitrogen Indirect Labelling Kit. The aaRNA samples were purified, lyophilized and eluted in 12 μl of DNAse/RNAse-free water prior to the dye coupling reactions. During dye labelling, samples were processed individually: DMSO was added to the Alexa dye tube, followed by aaDNA and incubated for 1h at room temperature. After a 1 h incubation at room temperature, the reactions were quenched using 4M hydroxylamine and the samples were purified, eluted in 35 μl of aaRNA elution buffer and quantified using a NanoDrop Spectrophotometer ND-1000. A standard reference design was used in the microarray experiments where the reference was comprised of a mixture of RNA from all individuals used in the study. All individual samples to be run on the arrays were fluorescently tagged with Alexa 555 and the mixed reference sample was labelled with Alexa 647. Prior to hybridization, dye labelled treatment and reference samples (825 ng each) were combined along with 1.2 μl 25X fragmentation buffer, 6 μl blocking agent and nuclease-free water to bring the final volume to 30 μl. The fragmentation mix was incubated at 60°C for 30 minutes and was stopped by adding 30 μl of 2X GEx hybridization buffer HI-RPM. Samples were briefly centrifuged, placed on ice and 55 μl of the mix was loaded into hybridization chambers in a Tecan-HS4800 Pro Hybridization Station.
|
| |
|
| |
| Hybridization protocol |
Each fish examined in the array experiments was run on a single slide against a reference control that was a pool of RNA from all of the fish used in the experiment. Data was normalized using the print-tip LOESS method. Microarray data were expressed in terms of normalized (background corrected) log2 ratios between each fish and the reference control. All slides were processed on Tecan-HS4800 Pro Hybridization Station (Tecan Trading AG, Switzerland). All steps from washing, hybridization, denaturation, and slide drying were carried out automatically.
|
| Scan protocol |
Fluorescent images were scanned using a Tecan LS Reloaded Scanner (Tecan Trading AG, Männedorf, Switzerland) and the analysis software Array-Pro Analyzer (Media Cybernetics, Inc., Bethesda, MD). The images were quantified using Imagene.
|
| Description |
raw data file (Ch1): Array2Ongo_09K_10380.txt
raw data file (Ch2): Array2Ongo_09K_10380ref.txt
|
| Data processing |
Expression data were managed using a local installation of BASE. BASE was customized slightly to support Imagene two-file formatting. Each slide was normalized in BASE using the print-tip LOESS method. The number of missing values, mean signal-to-noise log-ratio and quality metrics from arrayQualityMetrics and arrayQuality in Bioconductor were used to assess slide quality. Slides were removed from further experimental analysis if two or more plots were flagged on the arrayQualityMetrics report after data normalization, if more than 40% missing spots were identified by the SNR and missing spots report, if there were more than 30% missing spots and an experimentally low SNR value as identified by the SNR and missing spots report, or if the slide had spatial problems as identified by the plots from the arrayQuality package and substantiated by the spatial plots score of the raw microarray data in the arrayQualityMetrics report. The data for each retained slide was further processed to remove poor-quality spots. Flagged spots were treated as missing, as were spots with a SNR less than 2. In a set of slides considered a single data set, probes with more than 50% missing values were removed prior to statistical analysis. For procedures such as PCA that require matrices without missing values, missing data were imputed using an average of the existing probe intensities. The submitted data includes all probes and imputed data is not included.
|
| |
|
| Submission date |
Nov 27, 2012 |
| Last update date |
Nov 01, 2013 |
| Contact name |
Ken Jeffries |
| E-mail(s) |
[email protected]
|
| Organization name |
University of British Columbia
|
| Street address |
2424 Main Mall
|
| City |
Vancouver |
| State/province |
British Columbia |
| ZIP/Postal code |
V6T 1Z4 |
| Country |
Canada |
| |
|
| Platform ID |
GPL11299 |
| Series (2) |
| GSE42555 |
Transcriptomic responses to high water temperature in Pacific salmon [2007] |
| GSE42558 |
Transcriptomic responses to high water temperature in Pacific salmon |
|
