|
| Status |
Public on May 31, 2012 |
| Title |
Adams salmon 83594 held at 19.0C, Second |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
gill
|
| Organism |
Oncorhynchus nerka |
| Characteristics |
tissue: gill
alternative identifier: 83594
survival status: Survival
temperature (c): 19.0
stock: Adams
Sex: M
|
| 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 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 0.4 ml of TRI-reagent and one stainless steel 3 mm ball. Samples were 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 was Purity was assessed by measuring the A260/A280 ratio for purity and quantity. Total RNA (1 micrograms) was used as a template for RNA amplification using the Ambion MessageAmp II-96 aRNA Amplification Kit. Amplification steps were performed using one 96-well plate.
|
| Label |
Alexa 555
|
| Label protocol |
Amplified RNA (5 micrograms) was reverse transcribed into cDNA and labelled with Alexa dyes using the Invitrogen Indirect Labelling Kit, with modifications from the manufacturer's instructions. Briefly, the cDNA was purified using Zymo-25 Clean-Up columns (Zymo Research) and eluted using the 2X coupling buffer, supplied by Invitrogen. During dye labelling, samples were process individually: the DMSO was added to the Alexa dye tube, followed by cDNA and incubated for 1h at room temperature. 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. Labelled samples were cleaned up using Zymo-25 Clean-up columns by adding 300 microliters of DNA wash buffer to the pooled Alexa labelled cDNAs. The columns were centrifuged at 12500 rpm for 30 sec. The cDNA probes were washed 3 times with DNA Wash Buffer, centrifuged as above and re-centrifuged to dry followed by elution with 9 microliters of 1XTE buffer. The probes were combined with 2 microliters of Poly dA and denatured for 10 minutes at 80C. Following denaturation, 125 microliters of Ambion SlideHybe3 Buffer was added to the probes and held at 65 degrees C until the Tecan-HS4800 Pro Hybridization Station was ready for injection.
|
| |
|
| Channel 2 |
| Source name |
gill from all samples in the series
|
| Organism |
Oncorhynchus nerka |
| Characteristics |
tissue: gill
sample type: Pooled RNA reference
|
| Extracted molecule |
total RNA |
| Extraction protocol |
tbd
|
| Label |
Alexa 647
|
| Label protocol |
Amplified RNA (5 micrograms) was reverse transcribed into cDNA and labelled with Alexa dyes using the Invitrogen Indirect Labelling Kit, with modifications from the manufacturer's instructions. Briefly, the cDNA was purified using Zymo-25 Clean-Up columns (Zymo Research) and eluted using the 2X coupling buffer, supplied by Invitrogen. During dye labelling, samples were process individually: the DMSO was added to the Alexa dye tube, followed by cDNA and incubated for 1h at room temperature. 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. Labelled samples were cleaned up using Zymo-25 Clean-up columns by adding 300 microliters of DNA wash buffer to the pooled Alexa labelled cDNAs. The columns were centrifuged at 12500 rpm for 30 sec. The cDNA probes were washed 3 times with DNA Wash Buffer, centrifuged as above and re-centrifuged to dry followed by elution with 9 microliters of 1XTE buffer. The probes were combined with 2 microliters of Poly dA and denatured for 10 minutes at 80C. Following denaturation, 125 microliters of Ambion SlideHybe3 Buffer was added to the probes and held at 65 degrees C until the Tecan-HS4800 Pro Hybridization Station was ready for injection.
|
| |
|
| |
| 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 Perkin Elmer ScanArray Express, adjusting the PMT gain for optimized visualization of each image. The images were quantified using Imagene.
|
| 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 09, 2011 |
| Last update date |
May 18, 2016 |
| Contact name |
Paul Pavlidis |
| E-mail(s) |
paul@chibi.ubc.ca
|
| Organization name |
University of British Columbia
|
| Department |
Centre for High-Throughput Biology / Psychiatry
|
| Street address |
177 Michael Smith Laboratories 2185 East Mall
|
| City |
Vancouver |
| State/province |
British Columbia |
| ZIP/Postal code |
V6T 1Z4 |
| Country |
Canada |
| |
|
| Platform ID |
GPL2716 |
| Series (1) |
| GSE33586 |
Consequences of high temperatures and premature mortality on the transcriptome and blood physiology of wild adult sockeye salmon (Oncorhynchus nerka) |
|
