|
| Status |
Public on May 13, 2019 |
| Title |
LC-10h-A |
| Sample type |
RNA |
| |
|
| Source name |
Haemocyte cells, laboratory challenge, 10 hours, sample pool A
|
| Organism |
Ruditapes philippinarum |
| Characteristics |
tissue: haemocyte cells
sample type: Perkinsus olseni laboratory challenge
time point: 10h
pool: A
|
| Treatment protocol |
For LC experiment forty clams (40-45 mm shell length) were immersed individually in glasses containing 250 mL of filtered aerated seawater, either without parasite cells (control group) or with a suspension of 1 x 106 P.olseni zoospores (treatment group). The experiment lasted for 8 d and clams were not fed during this period.
|
| Growth protocol |
For WI study, 200 market-sized (≥ 40 mm in length, + 2 years old) adult Manila clams were collected from a P. olseni affected sea bed (Placeres, Ría de Pontevedra, NW Spain). In the case of the LC experiment, clams were collected from a P. olseni infection-free natural bed in Camariñas (Galicia, NW Spain) as confirmed by PCR (Casas et al., 2002a). These clams were kept at 14-17 oC in filtered (1 µm) seawater (35 ‰) with aeration for 1 week to adapt to indoor conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Haemolymph was collected from clam adductor muscle using a syringe with 25 mm gauge needle, and then transferred to a 1.5 mL tube and kept on crushed ice until haemocyte isolation. Haemolymph samples contaminated with bacteria or gametes were discarded after light microscope examination. Haemocytes were isolated from plasma by centrifuging haemolymph samples at 800 g for 10 min at 4 oC. The supernatant was then discarded and the pellet resuspended in 750 µL of RNAlater (Qiagen) solution and stored at -80 °C until RNA extraction. Total RNA was extracted using the Qiagen RNeasy mini kit with DNase following manufacturer’s instructions.
|
| 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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| |
|
| 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 (G2514F) 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.
|
| 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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| |
|
| Submission date |
May 22, 2017 |
| Last update date |
May 14, 2019 |
| 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 |
GPL23501 |
| Series (1) |
| GSE99162 |
Gene Expression Analysis of Ruditapes philippinarum Haemocytes after Experimental Perkinsus olseni Zoospore Challenge and Infection in the Wild |
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