|
| Status |
Public on Dec 01, 2012 |
| Title |
Pool b of gonads stage 3 females from Baie des Veys |
| Sample type |
RNA |
| |
|
| Source name |
Gonad pool, stage 3, females, BaiedesVeys
|
| Organism |
Magallana gigas |
| Characteristics |
individual/pool: pool
gender: female
tissue: gonad
Stage: 3
location: Locmariaquer
|
| Treatment protocol |
Oysters were sampled in the field and tissues were immediately dissected, snap-frozen in liquid nitrogen, and stored at -80 degrees Celsius for total RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the tissues with Tri-Reagent kit (Sigma) and subsequently cleaned with nucleospin RNA Clean-up (Macherev Nagel) isolation columns. After homogenization of the N2 grinding powder in Tri-reagent and the first centrifugation, the aqueous phase was extracted to directly isolate the total RNA on the column as described by the provider. RNA concentrations were measured using an ND-1000 spectrophotometer (Nanodrop Technologies), and RNA integrity was controlled on the Agilent Bioanalyzer using the RNA 6000 nano kit (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
200 ng of total RNA was indirectly labeled with Cy3 using the Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer's instructions. Qiagen's RNeasy mini spin columns were used for purifying amplified RNA (aRNA) samples. After purification, aRNA amplification and dye incorporation rates were controlled using a ND-1000 spectrophotometer (Nanodrop Technologies) and were comprised respectively between 200 and 500 ng/μL (concentration) and between 20 and 50 pmol/μg aRNA (dye incorporation).
|
| |
|
| Hybridization protocol |
Hybridization was performed using the Agilent’s Gene expression hybridization kit as described by the purchaser with 1.65μg of aRNA samples labeled with Cy3. Subsequently, slides were washed with Gene expression wash buffer kit (Agilent Technologies), dried and stabilized in Stabilization and Drying solution (Agilent Technologies).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565AA) using one color scan setting for 4x44k array slides (Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gc3 Fb
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 6.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. A matrix of gene expression levels was generated, where each row corresponds to a different gene and each column to one sample. The expression level of each gene was then logarithmically transformed, centered, and reduced, so that relative variations rather than absolute values were used for interpretation.
|
| |
|
| Submission date |
Dec 09, 2011 |
| Last update date |
Dec 01, 2012 |
| Contact name |
Nolwenn Marie Dheilly |
| E-mail(s) |
ndheilly@hotmail.fr
|
| Phone |
0625253514
|
| Organization name |
Universite de caen
|
| Department |
Biological sciences
|
| Lab |
PE2M
|
| Street address |
Esplanade de la Paix
|
| City |
Caen |
| ZIP/Postal code |
14000 |
| Country |
France |
| |
|
| Platform ID |
GPL11353 |
| Series (1) |
| GSE27955 |
Gametogenesis in an alternative hermaphrodite mollusk, the Pacific Oyster Crassostrea gigas: a microarray-based analysis identifies stage- and sex-specific genes |
|
