|
| Status |
Public on Oct 01, 2011 |
| Title |
PBS injected fish held at 10C prior to injection and sampled at 24 hours post injection - rep 6 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
PBS injected fish that were held at 10C prior to injection and sapled at 24 hours post injection - replicate 6
|
| Organism |
Gadus morhua |
| Characteristics |
tissue: Spleen
developmental stage: Naïve juveniles
population: New Brunswick Atlantic cod
treatment protocol: Held at 10°C and subsequently injected with PBS
|
| Treatment protocol |
At sampling time fish were euthanized by a lethal dose (0.4 g/l) of tricaine methanesulphonate and spleens were collected in RNAse-free 1.5 ml tubes, flash-frozen in liquid nitrogen and stored at -80°C until RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol (Invitrogen) following manufacturer's instructions. Total RNA was treated with DNAse I and column-purified using the RNeasy Mi-Elute Cleanup kit (Qiagen) following manufacturer's instructions.
|
| Label |
ALEXA 647
|
| Label protocol |
5 ug of DNAse-treated column-purified total RNA was labeled using Invitrogen SuperScript Direct cDNA Labeling kit according to the manufacturer's protocol. Test samples were always labeled with AlexaFluor 647, the common reference was always labeled with AlexaFluor 555.
|
| |
|
| Channel 2 |
| Source name |
Common Reference
|
| Organism |
Gadus morhua |
| Characteristics |
tissue: Spleen
developmental stage: Naïve juveniles
population: New Brunswick Atlantic cod
treatment protocol: Pool with equal contribution of RNA from every individual involved in the microarray experiment
|
| Treatment protocol |
At sampling time fish were euthanized by a lethal dose (0.4 g/l) of tricaine methanesulphonate and spleens were collected in RNAse-free 1.5 ml tubes, flash-frozen in liquid nitrogen and stored at -80°C until RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol (Invitrogen) following manufacturer's instructions. Total RNA was treated with DNAse I and column-purified using the RNeasy Mi-Elute Cleanup kit (Qiagen) following manufacturer's instructions.
|
| Label |
ALEXA 555
|
| Label protocol |
5 ug of DNAse-treated column-purified total RNA was labeled using Invitrogen SuperScript Direct cDNA Labeling kit according to the manufacturer's protocol. Test samples were always labeled with AlexaFluor 647, the common reference was always labeled with AlexaFluor 555.
|
| |
|
| |
| Hybridization protocol |
Arrays were prehybridized at 42°C in Nexterion Oligo Prehyb Buffer (Schott Nexterion cat. nr 1116889) for at least 2 hours. Arrays were washed at room temperature for with 0.1% SDS for 5 minutes, followed by 3 5-minute washes with water. Slides were dried by spinning them for 5 minutes at 200x g and kept at 42°C. LifterSlips were prepared by washing them sequentially with water, water, 70% ethanol and 100% ethanol and drying them using compressed air. For each array two labeled cDNAs were combined (one sample, one reference) and 2 μl of LNA blocker (Genisphere) and 40 μl of prewarmed 2x formamide-based hybridization buffer (Genisphere) was added. These samples were incubated at 80°C for 10 minutes and then kept at 42°C. Pre-warmed arrays were put in Corning hybridization chambers, LifterSlips were applied and the samples were loaded under the LifterSlips. Hybridizations were performed overnight for ~16 hours in a water bath at 42°C. After hybridization, arrays were washed at room temperature for 15 minutes with 2xSSC/0.2% SDS which was pre-warmed at 42°C. Next slides were washed at room temperature for 15 minutes with 2xSSC, followed by 2 15-minute washes with 0.2xSSC. Slides were dried by spinning them for 5 minutes at 200x g.
|
| Scan protocol |
Arrays were scanned at a resolution of 10 μm using a ScanArray Gx Plus scanner and ScanExpress v4.0 software (Perkin Elmer).
|
| Description |
Biological replicate 6 of 6. Control fish that were held at 10C for the duration of the experiment and subsequently injected with PBS.
|
| Data processing |
Signal intensity data was extracted from tiff images using Imagene v7.5. In addition to automated quality flagging, spots affected by dust particles, scratches and other local artefacts were flagged manually. Raw data was read into Bioconductor package 'marray', logratios were calculated from background-corrected median signals and control spots were removed. Data was normalized using printtip loess. Logratio data for a particular spot was removed when the raw signal value in one of the channels of that spot was lower than the threshold, which was calculated as (average background + 2* SD) for each channel on each array. Logratio data for a particular spot was also removed if the spot was flagged in Imagene. Logratios of duplicate spots were averaged.
|
| |
|
| Submission date |
Feb 14, 2011 |
| Last update date |
Oct 01, 2011 |
| Contact name |
Tiago Hori |
| E-mail(s) |
tshori@mun.ca
|
| Phone |
7098644794
|
| Organization name |
Memorial University of Newfoundland
|
| Department |
Ocean Sciences Centre
|
| Lab |
Rise Lab
|
| Street address |
0 Marine Lab Rd
|
| City |
St John's |
| State/province |
NL |
| ZIP/Postal code |
A1C5S7 |
| Country |
Canada |
| |
|
| Platform ID |
GPL10532 |
| Series (1) |
| GSE27299 |
The impact of chronic temperature elevation on the spleen anti-viral transcriptomic response in Atlantic cod (Gadus morhua) |
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