|
| Status |
Public on Feb 19, 2010 |
| Title |
PN24PV_bio2_tech2 |
| Sample type |
RNA |
| |
|
| Source name |
Pinot Noir leaf, Plasmopara viticola, 24h
|
| Organism |
Vitis vinifera |
| Characteristics |
tissue: leaf
infection: Plasmopara viticola
time point: 24h
cultivar: Pinot Noir
|
| Biomaterial provider |
Dr. Annalisa Polverari (University of Verona)
|
| Treatment protocol |
Fully expanded leaves of 8 to 10-week-old in vitro grapevine plants were infected by application of 50μL drops containing 50,000 sporangia per mL of Plasmopara viticola on the adaxial leaf surface. Leaf disks corresponding to infected tissue were cut and collected at 12 and 24 hours post-inoculation (hpi), immediately frozen in liquid nitrogen and stored at -80°C
|
| Growth protocol |
In vitro plants of Vitis vinifera cv. Pinot Noir grown at 27°C and a 16 h photoperiod (50 μE/m2/s) as described in Blaich (1977)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated form leaves of V. vinifera cv Pinot Noir according to Reid et al., 2006.
|
| Label |
Alexa Fluor 647
|
| Label protocol |
Total RNA (1 ug) was amplified by using the SuperScript Indirect RNA Amplification System (Invitrogen), to incorporate amino-allyl UTP molecules (aRNA) and fluorescently labelled by using the Alexa Fluor 647 reactive Dye
|
| |
|
| 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).
|
| Scan protocol |
Scanning was performed using Perkin Elmer ScanArray 4000XL microarray scanner and ScanArray Express 4.0 acquisition software. Laser was set at 60% of power. PMT gain was set at 60%.
|
| Description |
All the procedures provided were performed as indicated by Combimatrix protocols available at Combimatrix website (www.combimatric.com)
|
| Data processing |
Raw data was extracted using Microarray Imager 5.8.0 software (Combimatrix). Data were quantile-normalized using Blist 0.6 software (Combimatrix).
|
| |
|
| Submission date |
Oct 15, 2009 |
| Last update date |
Feb 19, 2010 |
| Contact name |
Alberto Ferrarini |
| E-mail(s) |
alberto.ferrarini@univr.it
|
| Phone |
+39-045-802-7058
|
| Organization name |
University of Verona
|
| Department |
Scientific and Technological Department
|
| Lab |
Plant Functional Genomics Centre
|
| Street address |
Strada le Grazie, 15
|
| City |
Verona |
| State/province |
Veneto |
| ZIP/Postal code |
37134 |
| Country |
Italy |
| |
|
| Platform ID |
GPL8025 |
| Series (1) |
| GSE18596 |
RNAs expression profiling in a resistant and a susceptible grapevine cultivar to Plasmopara viticola |
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