|
| Status |
Public on Apr 22, 2011 |
| Title |
June 20th Gills S |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
pool of 8 sensitive oyster gills, sampled the 20th June 2005
|
| Organism |
Magallana gigas |
| Characteristics |
sample: pool of 8 gills oysters
line: sensitive
field: in situ rearing (South Britanny, France)
gender: males and females
tissue: gills
|
| Biomaterial provider |
Laboratoire de Physiologie des Invertebres IFREMER Brest
|
| Treatment protocol |
in situ rearing before summer mortality (South Britanny, France)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Extract-All (Eurobio), RNA quality was assessed by Agilent 6000 nano reagents (Agilent), and RNA concentration was measured using an ND-1000 spectrophotometer (Nanodrop).
|
| Label |
Cy5
|
| Label protocol |
5µg of total RNA directly labeled by reverse transcription using the Direct ShipShot labeling kit (Promega).
|
| |
|
| Channel 2 |
| Source name |
pool of 10 wild oyster
|
| Organism |
Magallana gigas |
| Characteristics |
sample: pool of 10 oysters
line: no selection (wild oysters)
sampling: the 10th May 2007
field: Brest, France
gender: males and females
tissue: all tissues (whole oyster)
|
| Biomaterial provider |
Laboratoire de Physiologie des Invertebres IFREMER Brest
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Extract-All (Eurobio). RNA quality was assessed by Agilent 6000 nano reagents (Agilent). RNA concentration was measured using an ND-1000 spectrophotometer (Nanodrop).
|
| Label |
Cy3
|
| Label protocol |
5µg of total RNA directly labeled by reverse transcription using the Direct ShipShot labeling kit (Promega). All these Cy3 reference samples were pooled and then divided once more into several samples to obtain homogeneous reference.
|
| |
|
| |
| Hybridization protocol |
Equimolar amounts of cDNA samples and cDNA reference, labeled with Cy5 and Cy3 respectively, were SpeedVac evaporated and mixed into a single pool with the hybridization buffer (ChipHyb™ hybridization buffer, Ventana Discovery, Tucson, AZ, USA). They were then co-hybridized on the same microarray slide in a Ventana hybridization station (Ventana Discovery, Tucson, AZ, USA). Hybridizations were performed at the INRA IFR 140 transcriptomic facility (Rennes, France). Following pre-hybridization (with ChipSpread buffer, containing 4×SSC and 0.2% SDS), hybridization was conducted overnight at 42°C in a humidified chamber, according to manufacturer’s instructions. The arrays were washed twice with Ribowash solution (0.1 M Tris, 0.05 M EDTA, and 0.4 M NaCl), twice with 0.1×SSC and finally centrifuged to dry.
|
| Scan protocol |
After the hybridization step, microarray slides were scanned using a Scanner Genepix 4000B (Axon Instruments Inc.) according to the following parameters: Cy 5 Photo Multiplier Tube (PMT): 550 and Cy 3 PMT: 590.
|
| Data processing |
This process was repeated for each of the hybridized slides. The images (16-bits TIF images) were then analyzed with Genepix pro 5.1 software (Axon Instruments Inc.) according to manufacturer’s instructions. Spots were filtered for quality according to the following criteria: spot shape, median intensity of the spot, homogeneity of the local background pixels and spot pixel intensities. Spots showing inadequate signals were eliminated, as were those with noisy backgrounds and those with more than 20% saturated pixels in both channels. Transformation and normalization of hybridization data were performed to minimize variation arising from technical differences in RNA quality, probe labeling, and hybridization conditions between experiments. First, a logarithmic transformation was performed for each signal intensity (giving “log values”). The “log values” were median centered. Correction was next performed for differences in the variance across the range of gene expression levels using the formula: (corrected Cy5 log value)i = (Cy5 log value)i – (Cy3 log value)i + (mean Cy3 log value), where Cy5 log value represents sample signal intensity, Cy3 log value represents the reference signal intensity, and mean Cy3 log value represents the mean of all green values obtained for the gene “i” across the different conditions.
|
| |
|
| Submission date |
Nov 24, 2010 |
| Last update date |
Apr 23, 2011 |
| Contact name |
Arnaud Huvet |
| E-mail(s) |
ahuvet@ifremer.fr
|
| Phone |
+33 2 98 22 46 93
|
| Fax |
+33 (0)2 98 22 46 53
|
| Organization name |
IFREMER
|
| Department |
Physiologie Fonctionnelle des Organismes Marins
|
| Lab |
Laboratoire de Physiologie des Invertébrés
|
| Street address |
Ifremer centre de Brest BP 70
|
| City |
Plouzane |
| ZIP/Postal code |
29280 |
| Country |
France |
| |
|
| Platform ID |
GPL8639 |
| Series (1) |
| GSE25614 |
Microarray analysis highlights immune response of Pacific oysters as a determinant of resistance to summer mortality |
|
