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| Status |
Public on Dec 18, 2019 |
| Title |
H-ECP-1-R3 |
| Sample type |
RNA |
| |
|
| Source name |
Ruditapes philippinarum hemocytes cells-extracellular products in vitro challenge, 1 hour, replicate 3
|
| Organism |
Ruditapes philippinarum |
| Characteristics |
cell type: haemocyte cells
treatment: in vitro challenged with extracellular products from P. olseni, 1 hour
|
| Treatment protocol |
R. philippinarum hemocytes (5x106) were challenged in vitro with P. olseni trophozoites (5x106), zoospores (5x106) and extracellular products (2.5 mL of culture media enriched with extracellular products) separately in IWAKI 6-well plates. For the challenges, trophozoites and zoospores, obtained just before the challenge, were separately suspended in 2.5 mL filtered seawater (FSW) and added into a permeable insert (0.2 μm Anopore® membrane NUNC 25 mm) in each well. For hemocyte-extracellular products challenge, 2.5 mL of culture media enriched with hemocyte extracellular products were added into the inserts of the respective wells. The inserts allowed the flow of media but not the cells; hence, hemocytes and parasite cells were never in contact.
|
| Growth protocol |
Clams were collected from a P. olseni infection-free natural bed in Camariñas (Galicia, NW Spain) as confirmed by PCR (Casas et al., 2002a) and incubation of gill pieces in Ray’s fluid thioglycollate medium. These clams were kept at 14-17 oC in filtered (1 µm) seawater (35 ‰) with aeration for 1 week to adapt to indoor conditions. The details for collection of hemocytes have been described in Hasanuzzaman et al. (2017).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Hemocyte samples for RNA extraction were collected at 1, 8 and 24 h after the start of the challenge. 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.
|
| Description |
Gene expression of Ruditapes philippinarum hemocytes in vitro challenged with extracellular products from P. olseni, 1 hour, replicate 3
|
| 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 |
Dec 17, 2019 |
| Last update date |
Dec 18, 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 (2) |
| GSE142172 |
Manila clam - Perkinsus olseni interaction based on gene expression analysis of clam hemocytes and parasite trophozoites [Manila clam] |
| GSE142666 |
New insights into the Manila clam – Perkinsus olseni interaction based on gene expression analysis of clam hemocytes and parasite trophozoites through in vitro challenges |
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