|
| Status |
Public on Sep 01, 2014 |
| Title |
534330A07 - 12_TREATMENT3_48H_REP1 vs 8_WATER_48H_REP1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
8_WATER_48H_REP1
|
| Organism |
Vitis vinifera |
| Characteristics |
variety: cv. Marselan
age: 6week
tissue: herbaceous grape cutting
|
| Treatment protocol |
Name:WATER_48H - compound based treatment - pathogen infection,water:.
|
| Growth protocol |
caulin leaf - Growth conditions (Media/hygrometry/Temperature/Light) -peat+perlite (4:1). RH 65%. 20+/-2°C. Daylight + HQI light
|
| Extracted molecule |
total RNA |
| Extraction protocol |
8_WATER_48H_REP1:80ug.
|
| Label |
Cy5
|
| Label protocol |
labelling Cy3 and Cy5 direct, amplification=yes, aRNA 5 ug. (Labelling_protocol.txt) Labelling protocol: 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).
|
| |
|
| Channel 2 |
| Source name |
12_TREATMENT3_48H_REP1
|
| Organism |
Vitis vinifera |
| Characteristics |
variety: cv. Marselan
age: 6week
tissue: herbaceous grape cutting
|
| Treatment protocol |
Name:T3_48H - compound based treatment - pathogen infection,water inoculation:.
|
| Growth protocol |
caulin leaf - Growth conditions (Media/hygrometry/Temperature/Light) -peat+perlite (4:1). RH 65%. 20+/-2°C. Daylight + HQI light
|
| Extracted molecule |
total RNA |
| Extraction protocol |
12_TREATMENT3_48H_REP1:80ug. (Reid_2006_RNA_extra.pdf)
|
| Label |
Cy3
|
| Label protocol |
labelling Cy3 and Cy5 direct, amplification=yes, aRNA 5 ug. (Labelling_protocol.txt) Labelling protocol: 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).
|
| |
|
| |
| Hybridization protocol |
8_WATER_48H_REP1 Cy5 / 12_TREATMENT3_48H_REP1 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.
|
| Scan protocol |
Mapix, Cy3:pmt voltage 532nm,480V,laser power 35%, Cy5:635nm,pmt voltage 480V,laser power 30%
|
| Description |
Biological question (15 lines max): Identification of grape genes regulated in response to resistance inducer treatments. This transcriptomic analysis should provide insight in the mode of action of the compounds under study and yield generic data relevant to grape defense reaction.
|
| Data processing |
For each array, the raw data comprised the logarithm of median feature pixel intensity at wavelengths Cy5 (red) and Cy3 (green).For each array, a global intensity-dependent normalization using the loess procedure (Yang et al., 2002) was performed to correct the dye bias.Log-ratios are then averaged over the duplicate probes to get a value per gene.
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| |
|
| Submission date |
Jan 23, 2014 |
| Last update date |
Sep 01, 2014 |
| Contact name |
Stéphanie Pateyron |
| E-mail(s) |
pateyron@evry.inra.fr
|
| Organization name |
IPS2_Institute of Plant Sciences Paris-Saclay
|
| Lab |
Transcriptomic Plateforme POPS
|
| Street address |
Rue de Noetzlin _ Batiment 630
|
| City |
Orsay |
| ZIP/Postal code |
91405 |
| Country |
France |
| |
|
| Platform ID |
GPL18213 |
| Series (2) |
| GSE54345 |
12plex_vitis_2011_02_48h |
| GSE54347 |
Identification of grape genes regulated in response to resistance inducer treatments |
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