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| Status |
Public on Jul 27, 2018 |
| Title |
Limfjord, haemocytes, 90 days, Bonamia, sample 7 |
| Sample type |
RNA |
| |
|
| Source name |
Limfjord_haemocyte cells_90 days_Bonamia
|
| Organism |
Ostrea edulis |
| Characteristics |
strain: Limfjord
phenotype: naïve (NS)
tissue: haemocyte cells
challenged with: B. ostreae cells
sampling time point: 90 day
|
| Treatment protocol |
The challenge was performed by individually immersing oysters in beakers containing an aerated suspension of 300,000 B. ostreae cells in filtered seawater for 24 h at the Centro de Investigacións Mariñas. The B. ostreae cells for the challenge were isolated from heavily infected oysters collected in Lough Foyle (Ireland), following the procedure described by Mialhe et al. (1988). After challenge, oysters were kept in tanks with running, filtered (1 µm) seawater plus continuous (pumped) supply of mixed cultured algae until the end of the experiment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Haemolymph samples were taken at three different times after the challenge (1, 30 and 90 days post-challenge, dpc); as much haemolymph as possible (from 0.2 ml to 1.5 ml) was withdrawn from the adductor muscle of each oyster with a 21 gauge needle screwed to a 2 ml cold syringe. Immediately the haemolymph was poured into a cold vial and kept in crushed ice to avoid haemocyte clumping. Haemolymph samples were centrifuged (800 g, 10 min, 4ºC) and the cell fractions (pellets) were resuspended in RNA later and stored at -20ºC after one night at 4ºC until RNA extraction. RNA was individually extracted by RNeasy Mini Kit (Qiagen, Germany) following the manufacturer's instructions with slight modifications, including two additional RPE washes and the optional DNase digestion.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng RNA of each strain and time using the Low Input Quick Amp Labeling Kit, One-Color (Cy3) (Agilent Technologies, USA) 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.
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| |
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| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >6 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent Hi-RPM hybridization buffer was added to the fragmentation mixture and hybridized to turbot custom Oligo Microarrays (G2509F) 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).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565B) using one color scan setting for 8x15k array slides.
|
| Description |
L1T3B-7
Limfjord, gene expression at 90 days after Bonamia ostreae infection sample 7
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5 (Agilent) using default parameters (protocol GE1-v5_95) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Extended dynamic range implemented in the Agilent software was applied to avoid saturation in the highest intensity range. The Agilent feature extraction was used as raw data for further preprocessing. The processed signal (gProcessed-Signal) value was chosen for the statistical analysis.
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| |
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| Submission date |
Aug 09, 2017 |
| Last update date |
Jul 27, 2018 |
| Contact name |
Belen G Pardo |
| E-mail(s) |
belen.gomez@usc.es
|
| Phone |
+34 982822428
|
| Organization name |
University of Santiago de Compostela
|
| Department |
Zoology, Genetics and Physical Anthropology
|
| Lab |
Acuigen
|
| Street address |
Avda. Carballo Calero s/n
|
| City |
LUGO |
| State/province |
LUGO |
| ZIP/Postal code |
27002 |
| Country |
Spain |
| |
|
| Platform ID |
GPL23881 |
| Series (1) |
| GSE102405 |
Differential gene expression between long-term affected and naïve flat oyster (Ostrea edulis) stocks in response to Bonamia ostreae |
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