|
| Status |
Public on Aug 04, 2009 |
| Title |
Spleen 72h infected vs. uninfected 8 (2C) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Spleen 72h 8
|
| Organism |
Scophthalmus maximus |
| Characteristics |
tissue: spleen
infection: uninfected control
|
| Growth protocol |
40 individuals (20-30 g each) obtained from a mixture of heterogeneous genetic families was collected at CETGA (Aquaculture Galician Technological Centre)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from pooled tissues of control and infected fish using TRIZOL Reagent (Life Technologies) according to manufacturer’s recommendations. All extractions were performed by the same researcher.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) and Cyanine-5 (Cy5) labeled cRNA was prepared from 50 ng RNA using the Two-Color Microarray-Based Gene Expression Analysis (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
Spleen 72h 8
|
| Organism |
Scophthalmus maximus |
| Characteristics |
tissue: spleen
infection: Aeromonas salmonicida infected
|
| Growth protocol |
40 individuals (20-30 g each) obtained from a mixture of heterogeneous genetic families was collected at CETGA (Aquaculture Galician Technological Centre)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from pooled tissues of control and infected fish using TRIZOL Reagent (Life Technologies) according to manufacturer’s recommendations. All extractions were performed by the same researcher.
|
| Label |
Cy5
|
| Label protocol |
Cyanine-3 (Cy3) and Cyanine-5 (Cy5) labeled cRNA was prepared from 50 ng RNA using the Two-Color Microarray-Based Gene Expression Analysis (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
300 ng ug of each Cy3 and Cy5-labelled cRNA (specific activity >8.0 pmol Cy3-Cy5/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul adding 1ul 25x Agilent fragmentation buffer and 5 ul 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction 25 ul 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to the turbot custom oligo-microarray for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using two color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5 um).
|
| Description |
Gene expression afer challenging with Aeromonas salmonicida
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5 (Agilent) using default parameters (protocol GE2-v5_95_Feb07 and Grid:016528_D_F_20070525) to obtain Processed Signal intensities. Those microarrays having an average coefficient of variation (CV) across Spike-In and non-Spike-In probes below 10% were considered for further analysis. The Spike-Ins comprise 10 in vitro synthesized, 20-fold replicated polyadenylated transcripts, which are premixed at 10 concentration ratios to monitor the system for linearity, sensitivity and accuracy across the intensity range. Secondly, the following features and/or genes which did not conform to the established quality criteria were filtered: i) non-uniform pixel distributed outliers and population replicate outliers according to the default Agilent feature extraction criteria; ii) features whose ratio between processed signal and its error was below 2; iii) spots not differentiated from background signal (as estimated for each spot); iv) features below the limit where the linear relationship between concentration and intensity was lost according to Spike-In information; v) genes which did not show at least 3 quality replicates out of 5 and/or a coefficient of variation (CV) across replicates >10%.
|
| |
|
| Submission date |
Jul 27, 2009 |
| Last update date |
Aug 03, 2009 |
| Contact name |
Adrian Millan Perez |
| E-mail(s) |
adrian.millan@usc.es
|
| Organization name |
Universidad Santiago Compostela
|
| Department |
Acuigen
|
| Street address |
Facultad Veterinaria, Campus Universitario Pabellon IV 2 superior
|
| City |
Lugo |
| State/province |
Lugo |
| ZIP/Postal code |
27002 |
| Country |
Spain |
| |
|
| Platform ID |
GPL8937 |
| Series (1) |
| GSE17357 |
Design and performance of a turbot (Scophthalmus maximus) oligo-microarray based on ESTs from immune tissues |
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