|
| Status |
Public on Dec 27, 2019 |
| Title |
TP-C-R4 |
| Sample type |
RNA |
| |
|
| Source name |
Perkinsus olseni trophozoites in vitro challenge, control pool, replicate 4
|
| Organism |
Perkinsus olseni |
| Characteristics |
tissue: trophozoites
treatment: in vitro challenge with filtered seawater, control pool
|
| Treatment protocol |
P. olseni trophozoites were in vitro challenged with 2.5 mL of plasma (treatment) or FSW (control), added into a permeable insert (0.2 µm Anopore® membrane NUNC 25 mm) set in the plate-wells. Four pseudo-replicates (trophozoites from the same culture) for both control and treatment groups were collected at 1, 8 and 24 h since the onset of the challenge.
|
| Growth protocol |
P. olseni trophozoites were obtained from the gills of parasitized R. decussatus clams, and in vitro cultured for 1–2 months following the procedure described by Casas et al. (2002). A volume of culture containing ~5x10e6 trophozoites was taken from each culture and centrifuged (1000g, 25C, 10 min) to concentrate cells. The concentrated trophozoites were resuspended in 2.5 mL filtered sea water and added to IWAKI 6-well plates.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
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 RNeasy 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 manufacturer's 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). A single pooled control was hybridized in two microarrays per slide as technical replicates. Twelve microarrays were used for the treatments across the time-series (1 h, 8 h and 24 h) including four replicates per time point.
|
| 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 Perkinsus olseni trophozoites in vitro challenge, control pool replicate 4.
|
| 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 27, 2019 |
| Last update date |
Dec 27, 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 |
GPL27953 |
| Series (2) |
| GSE142665 |
New insights into the Manila clam – Perkinsus olseni interaction based on gene expression analysis of clam hemocytes and parasite trophozoites through in vitro challenges [Perkinsus olseni] |
| 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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