|
| Status |
Public on Dec 03, 2019 |
| Title |
Coho smolt Brain_3712 [56105] |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
brain
|
| Organism |
Oncorhynchus kisutch |
| Characteristics |
FISH #: 3712
fw/sw: SW
year: 2009
season: Ocean_Fall
origin: Hatchery
fork length (cm): 31.4
total mass (g): 385
Sex: M
latitude: 50.745
longitude: -127.134
spatial1: WCVI
spatial2: WCVI
region1: ECVI
region2: ECVI
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from four tissues separately: brain, gill, muscle, and liver. Tissues (10mg) were homogenized in TRI reagent (Ambion) using stainless steel beads on a MM301 mixer mill (Retsch Inc.). Aqueous layer homogenate aliquots (100?L) were transferred to three U-bottom 96 well plates. RNA was extracted from the homogenates using the ÔNo-Spin ProcedureÕ of MagMAX-96 for Microarrays Total RNA Isolation kits (Ambion) and a Biomek NXP automation workstation (Beckman- Coulter). RNA yield and purity were measured by spectrophotometry (A?260 value or A?260/A?280 ratio). RNA extracts were stored in a ?80¡C freezer.
|
| Label |
Alexa 555
|
| Label protocol |
RNA extracts were amplified for microarray hybridizations using Amino Allyl MessageAmpª II-96 kits (Ambion). Amplified RNA extracts (5?g, aRNA) were labelled with Alexa dyes using Indirect Labelling kits (Invitrogen). Dye coupling reactions were performed using all aRNA samples with Alexa 555 dye and the reference aRNA (a pool of all samples for a tissue) with Alexa 647.
|
| |
|
| Channel 2 |
| Source name |
brain from all samples in the series
|
| Organism |
Oncorhynchus kisutch |
| Characteristics |
sample type: Pooled cRNA reference
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from four tissues separately: brain, gill, muscle, and liver. Tissues (10mg) were homogenized in TRI reagent (Ambion) using stainless steel beads on a MM301 mixer mill (Retsch Inc.). Aqueous layer homogenate aliquots (100?L) were transferred to three U-bottom 96 well plates. RNA was extracted from the homogenates using the ÔNo-Spin ProcedureÕ of MagMAX-96 for Microarrays Total RNA Isolation kits (Ambion) and a Biomek NXP automation workstation (Beckman- Coulter). RNA yield and purity were measured by spectrophotometry (A?260 value or A?260/A?280 ratio). RNA extracts were stored in a ?80¡C freezer.
|
| Label |
Alexa 647
|
| Label protocol |
RNA extracts were amplified for microarray hybridizations using Amino Allyl MessageAmpª II-96 kits (Ambion). Amplified RNA extracts (5?g, aRNA) were labelled with Alexa dyes using Indirect Labelling kits (Invitrogen). Dye coupling reactions were performed using all aRNA samples with Alexa 555 dye and the reference aRNA (a pool of all samples for a tissue) with Alexa 647.
|
| |
|
| |
| Hybridization protocol |
Gene expression was quantified using 44K cGRASP salmonid microarrays (Koop et al., 2008) for each of the four tissues. Fragmented sample and reference cRNA (dye coupled aRNA) in GEx Hybridization HI-RPM buffer (Agilent) were hybridized to microarray slides using aHS4800 Pro Hybridization Station (TecanTrading AG), which automatically washed, hybridized, denatured, and dried the slides. To reduce technical variance, hybridizations were performed by a single technician in batches of up to 48 samples during a two-week period.
|
| Scan protocol |
Slides were read using a LS Reloaded scanner (TecanTrading AG) and Array- Pro Analyzer software. Images were quantified using ImaGene software (BioDiscovery) with spots being automatically flagged if both dye wavelengths were empty, poor, or negative and manually flagged for reasons such as filaments, blobs, or smears.
|
| Description |
Sample array position within slide:
Array3Onkism0809_B_10128
raw data files:
Onkism0809_B_10128.txt
Onkism0809_B_10128_ref.txt
sample name in processed data file:
56105
*ref.txt file is the raw data for channel 2 (pooled reference.
|
| Data processing |
Low quality slides (>50% missing spots or spatial problems) and spots (<2 standard deviations from the mean background at both dye wavelengths) were removed using BASE software (Lund University). Local background correction using background mean and a lowess normalization algorithm were also applied.
The file processed_data_brain_withallcontrols.txt includes normalized log2 background-subtracted intensity ratio (each fish/reference) with 'flagged' spots or poor signals removed for all probes including controls. Sample data tables do not include control probes.
|
| |
|
| Submission date |
Dec 03, 2018 |
| Last update date |
Dec 03, 2019 |
| 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 |
GPL11299 |
| Series (2) |
| GSE123304 |
Transcriptional shifts during juvenile Coho salmon (Oncorhynchus kisutch) life stage changes in freshwater and early marine environments [brain] |
| GSE123308 |
Transcriptional shifts during juvenile Coho salmon (Oncorhynchus kisutch) life stage changes in freshwater and early marine environments |
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