|
| Status |
Public on Jan 24, 2012 |
| Title |
Liver control |
| Sample type |
RNA |
| |
|
| Source name |
Liver control
|
| Organism |
Scophthalmus maximus |
| Characteristics |
time point: time 0; non-injected
tissue: liver
|
| Treatment protocol |
Fish were injected intracelomically (i.p.) with 0.1 ml of a parasite suspension of 2.5x10^6 parasites/mL. The presence of P. dicentrarchi in infected fish was checked by plating drops of the ascitic fluid and blood that were impregnated with ammoniacal pyridinated silver carbonate, and biometric measurements of taxonomically important characters were made under an optical microscope (Budiño et al., 2011b). The challenges were performed at the CETGA facilities in quarantine tanks. All fish used in the experiment were anaesthetized with benzocaine (50 mg/ml) and sacrificed by decapitation. Equal amounts of spleen, liver and head kidney were sampled from each fish, pooled per organ at each sampling point and immediately stored in liquid nitrogen.
|
| Growth protocol |
150 individuals of ~ 30g from 20 equally represented families from a turbot company (Alrogal SA) were transported to CETGA (Centro Tecnológico Gallego de Acuicultura; NW Spain) facilities. Prior to challenge, fry were maintained during 10 days at standard culture conditions to cut down on transport stress and to check if they were free from diseases.
|
| 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) labeled cRNA was prepared from 0.2 ug RNA using the One-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 |
0,6 ug of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/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 one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5 um).
|
| Description |
Gene expression after challenging with Philasterides dicentrarchi
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5 (Agilent) using default parameters (protocol GE1-v5_95 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. 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 |
Jan 18, 2012 |
| Last update date |
Jan 24, 2012 |
| 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) |
| GSE35184 |
Gene expression profiles of spleen, liver and head kidney in turbot (Scophthalmus maximus) along the infection process with Philasterides dicentrarchi using an immune-enriched oligo-microarray |
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