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Status |
Public on Apr 28, 2011 |
Title |
21/66_12PV_bio2_tech1 |
Sample type |
RNA |
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Source name |
F1 21/66_infected_12h
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Organism |
Vitis vinifera |
Characteristics |
genotype: F1 21/66 (Merzling x Teroldego cross) tissue: leaf infection: Plasmopara viticola time point: 12h
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Treatment protocol |
Sporangiophores of P. viticola (Berk. and Curt) Berl. et De Toni were collected from infected leaves of V. vinifera cv Pinot Gris plants by brushing the white mould present on the underside of the leaves in cold bidistilled water. Fully expanded leaves of 8 to 10 week old grafted plants were inoculated by spraying a conidial suspension of 10^4/10^5 spores/ml onto the abaxial leaf surface and were kept overnight in the dark in a growth chamber at 24°C with 80% RH . The infected plants were then transferred to the greenhouse and kept in the same conditions as described above. Mock-inoculated plants were obtained by spraying distilled water in the greenhouse. Leaves for the analysis (the second and third from the apex) were collected from two biological replicates each of F1 21/66, F1 22/73 and Teroldego at 12 and 96 hours post infection (hpi) and at 0 hours post mock-inoculation (hpmi), immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction.
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Growth protocol |
F1 individuals from Merzling x Teroldego cross were replicated annually by grafting wood cuttings onto rootstock KOBER 5BB. The plants were kept in a growth chamber in 1L pots filled with soil:sand:peat:vermiculite (3:1:3:3, v/v) in greenhouse at 25°C/20°C day/night temperature, 16 h photoperiod and a relative humidity (RH) of 70 ± 10%.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted from frozen leaves according to Moser et al., 2004
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Label |
Cy5
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Label protocol |
Total RNA (1 microgram) was amplified using the Amino Allyl MessageAmpII aRNA Amplification kit (Ambion, USA) and the resulted amminoallyl-aRNA was conjugated to a fluorescent label (Cy-5). The purified labeled aaRNA was quantified by spectrophotometry (ATI Unicam) and 2 micrograms were hybridized to the custom Combimatrix array according to the manufacters directions.
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Hybridization protocol |
Prehybridization was performed by incubating the arrays with prehybridization solution (6X SSPE, 1% SDS, 0.2% BSA) for 60 minutes at 37 °C. Hybridization was performed at 37°C for 16 hours in hybridization solution (6X SSPE, 0.8% BSA, 12% DI Formamide, 2.5% SDS). Hybridization washings were performed as following: - wash with 6X SSPET wash solution (6X SSPE, 0.05% Tween-20). - wash with 3X SSPET wash solution (3X SSPE, 0.05% Tween-20). - 2 washes with 0.5X SSPET wash solution (0.5X SSPE, 0.05% Tween-20).
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Scan protocol |
The arrays were scanned with a ScanArray4000XL (Perkin Elmer, USA) and TIF images were exported to MicroArray Imager 5.8 (Combimatrix, USA) for densometric analysis.
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Description |
21/66_12PV_D1
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Data processing |
Raw data was extracted using Microarray Imager 5.8.0 software (Combimatrix). Spot flagging and visual inspection of the images was carried out in order to exclude bad spots (based on spot saturation and heterogeneity). The raw signals were analyzed and negatively flagged spots were excluded from further analysis by assigning them a zero weight. Only probes with a signal intensity of at least 500 fluorescence units (Galbraith, 2003) for all biological replicates were considered for further analysis. A scaling normalization was further performed using Actin and Ufgt (UDP-glucose:flavonoid 3-O-glucosyltransferase) as reference genes. A mean expression value from normalized values of technical replicates for each probe was calculated.
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Submission date |
Apr 26, 2011 |
Last update date |
Aug 18, 2011 |
Contact name |
Giulia Malacarne |
E-mail(s) |
giulia.malacarne@fmach.it
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Phone |
+39-0461-615387
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Organization name |
Edmund Mach Foundation
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Department |
Genomics and Biology of Fruit Crops Department
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Street address |
Via Edmund Mach,1
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City |
S. Michele a/Adige |
State/province |
Trento |
ZIP/Postal code |
38010 |
Country |
Italy |
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Platform ID |
GPL13450 |
Series (1) |
GSE28851 |
Resistance to Plasmopara viticola in a grapevine segregating population is associated with stilbenoid accumulation and with specific host transcriptional responses |
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