|
| Status |
Public on Nov 05, 2010 |
| Title |
Spleen_PBS_24HPI_fish3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Spleen tissue, PBS group, 24 hours post-injection
|
| Organism |
Gadus morhua |
| Characteristics |
tissue: Spleen
life stage: Juvenile
agent: 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 |
AlexaFluor 647
|
| Label protocol |
5 μg 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 |
Pooled total RNA from spleen tissue, undisturbed control group
|
| Organism |
Gadus morhua |
| Characteristics |
tissue: Spleen
life stage: Juvenile
sample type: Pooled total RNA from spleen tissue, undisturbed control group
|
| 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 |
AlexaFluor 555
|
| Label protocol |
5 μg 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 5 μm using a ScanArray Gx Plus scanner and ScanExpress v4.0 software (Perkin Elmer).
|
| Description |
n/a
|
| 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', base 2 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 |
Jun 11, 2010 |
| Last update date |
Jan 21, 2014 |
| Contact name |
Marije Booman |
| E-mail(s) |
[email protected]
|
| Organization name |
Memorial University of Newfoundland
|
| Department |
Ocean Sciences
|
| Street address |
1 Marine Lab Road
|
| City |
St. John's |
| State/province |
NL |
| ZIP/Postal code |
A1C 5S7 |
| Country |
Canada |
| |
|
| Platform ID |
GPL10532 |
| Series (1) |
| GSE22312 |
The study of Atlantic cod (Gadus morhua) spleen global gene expression response to intraperitoneal injection with formalin-killed atypical Aeromonas salmonicida. |
|
