|
| Status |
Public on Dec 10, 2010 |
| Title |
S. salar_89_dpf_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Whole tissue, 89 days post fertilization
|
| Organism |
Salmo salar |
| Characteristics |
tissue: whole organism
developmental stage: 89 days post fertilization
|
| Growth protocol |
Eggs from Atlantic salmon (McConnell (Mowi)) were obtained in November, 2009 from Marine Harvest United Hatchery (Fanny Bay, B.C., Canada). The eggs were fertilized by gently mixing the eggs and milt by hand and then washed with partial exchanges of water. Approximately 2000 fertilized eggs were then transferred and placed in Heath trays (Marisource) at the University of Victoria. The embryos and larvae were raised in fresh water at a temperature of 12°C and a flow rate of 200 liters/h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted in TRIzol reagent (Invitrogen) by mixer-mill homogenization (Retsch) and spin-column purified using RNeasy Mini kits (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
Cy5 labeled experimental cRNA samples was generated using an Agilent Low Input Quick Amp (LIQA) kit, following the manufacturer’s instructions. For each time point, 40 ng of total RNA from three individuals was used to generate first-strand cDNA. Agilent Spike-In B control RNA was included in each reaction. After the denaturation step (10 min at 65 ºC) and cRNA synthesis step (2 hr at 40 ºC), the reactions were incubated at 70 ºC for 15 minutes to inactivate the AffinityScript enzyme, and subsequently stored at -80 ºC until further use. For the labeling reactions, thawed cRNA samples were each mixed with 16 µL of Transcription Master Mix cocktail containing Cy5 dye, and incubated at 40 ºC for two hours. Purification was performed using Qiagen RNeasy mini spin columns, eluting in 30 µL of RNase-free water. For the generation of the reference pool, equimolar amounts from the three individuals in each time point were pooled to give 120 ng of total RNA used in each first-strand reaction. Spike-In A control RNA was included in each reaction. After labeling with Cy3 and column purification as above, a common reference pool was created by including 2.8 µg of cRNA from each time point, except for D2, for which only 1.3 µg of material was available due to the small size of the animal at this stage.
|
| |
|
| Channel 2 |
| Source name |
Pooled cRNA from whole tissues in all time points
|
| Organism |
Salmo salar |
| Characteristics |
tissue: whole organism
sample type: Pooled cRNA from whole tissues in all time points
|
| Growth protocol |
Eggs from Atlantic salmon (McConnell (Mowi)) were obtained in November, 2009 from Marine Harvest United Hatchery (Fanny Bay, B.C., Canada). The eggs were fertilized by gently mixing the eggs and milt by hand and then washed with partial exchanges of water. Approximately 2000 fertilized eggs were then transferred and placed in Heath trays (Marisource) at the University of Victoria. The embryos and larvae were raised in fresh water at a temperature of 12°C and a flow rate of 200 liters/h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted in TRIzol reagent (Invitrogen) by mixer-mill homogenization (Retsch) and spin-column purified using RNeasy Mini kits (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
Cy5 labeled experimental cRNA samples was generated using an Agilent Low Input Quick Amp (LIQA) kit, following the manufacturer’s instructions. For each time point, 40 ng of total RNA from three individuals was used to generate first-strand cDNA. Agilent Spike-In B control RNA was included in each reaction. After the denaturation step (10 min at 65 ºC) and cRNA synthesis step (2 hr at 40 ºC), the reactions were incubated at 70 ºC for 15 minutes to inactivate the AffinityScript enzyme, and subsequently stored at -80 ºC until further use. For the labeling reactions, thawed cRNA samples were each mixed with 16 µL of Transcription Master Mix cocktail containing Cy5 dye, and incubated at 40 ºC for two hours. Purification was performed using Qiagen RNeasy mini spin columns, eluting in 30 µL of RNase-free water. For the generation of the reference pool, equimolar amounts from the three individuals in each time point were pooled to give 120 ng of total RNA used in each first-strand reaction. Spike-In A control RNA was included in each reaction. After labeling with Cy3 and column purification as above, a common reference pool was created by including 2.8 µg of cRNA from each time point, except for D2, for which only 1.3 µg of material was available due to the small size of the animal at this stage.
|
| |
|
| |
| Hybridization protocol |
cRNA fragmentation mixtures were created following the LIQA kit instructions, using 825 ng of experimental sample and 825 ng of reference pool. These mixtures were incubated at 60 ºC for 30 minutes. After cooling on ice for one minute, hybridization mixtures were prepared by adding 2x GEx Hybridization Buffer HI-RPM and mixing well by pipetting. These reactions were loaded in random arrangements with respect to time point onto 44K oligo salmonid microarrays (Agilent-025055) using Agilent SureHyb Hybridization Chambers. Each of the 4x44K arrays on the microarray slides had 100 uL of hybridization reaction added. The hybridization reactions were allowed to occur for 17 hours at 65 ºC. Slide washes were performed as per the manufacturer’s instructions, including an ozone-protection step using the Agilent Stabilization and Drying Solution.
|
| Scan protocol |
Slides were scanned as soon as possible on a ScanArray Express (Perkin Elmer) scanner at 5 µm resolution using a PMT setting of 80 in both channels, a black threshold of 1800, and a full color threshold of 26.8. Slides were stored in a low ozone chamber (typically <5 ppb) until scanned.
Images were quantified using Imagene 8.0 (Biodiscovery).
|
| Description |
Biological replicate 1 of 3
|
| Data processing |
Background corrected signal medians converted to threshold of 1.0, log2 transformed, Lowess normalized, baseline transformation to median.
|
| |
|
| Submission date |
Dec 08, 2010 |
| Last update date |
Dec 10, 2010 |
| Contact name |
Ben F Koop |
| E-mail(s) |
bkoop@uvic.ca
|
| Phone |
(250) 472-4067
|
| Organization name |
The University of Victoria
|
| Department |
Biology
|
| Lab |
Centre for Biomedical Research
|
| Street address |
PO Box 3020 STN CSC
|
| City |
Victoria |
| State/province |
BC |
| ZIP/Postal code |
V8W 3N5 |
| Country |
Canada |
| |
|
| Platform ID |
GPL11299 |
| Series (1) |
| GSE25938 |
Developmental stages after fertilization in Salmo salar |
|
