|
| Status |
Public on Jul 26, 2019 |
| Title |
control_T1_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
berry pericarp water-injected T1 (5 days after injection), replicate 3
|
| Organism |
Vitis vinifera |
| Characteristics |
cultivar: Muller-Thurgau
tissue: Berry pericarps
type of treatment: water-injected
|
| Treatment protocol |
Among the sterilized berries, 200 berries were frozen, divided into three different biological replicates (a pool of 65 berries each) and sampled as time point 0 (T0) of the experiment. Botrytis cinerea B05.10 was cultured and inoculated into 400 berries (1x105 conidia per ml). The remaining 400 berries were water-injected and used as control. Berries were incubated into sterile 24-well plates under controlled conditions (15°C/15h dark; 18°C/9h light) to induced noble rot. Three biological replicates (about 50 berries each) of infected/control berries were collected and frozen 5 days after the injection when inoculated berries were at the initial stage of noble rot development (time point 1, T1). Moreover, infected/control berries were sampled 10 days after the injection when the inoculated berries were at the pourri plain stage of noble rot (time point 2, T2) characterized by a complete and homogeneous chocolate hue.
|
| Growth protocol |
Vitis vinifera cv. Muller-Thurgau bunches were harvested in August 2017 in Custoza (Italy) and dehydrated into the withering setting of the “La Prendina” farm located in Monzambano (Mantova, Italy). Muller-Thurgau grapes were collected with a soluble solids content of 18.25 ± 0.05 °Brix and placed into plateaux (about 20 kg each) in a ventilated withering facility (14-15°C, 53-60% of relative humidity). Brix degree and weight loss of berry bunches where monitored weekly during withering. After one month, berry Brix degree was about 24 and the percentage of weight loss was ~30% of the initial weigh. One thousand Muller-Thurgau berries (with pedicels) were sampled, surface sterilized for 5 minutes in 70% Ethanol and dried in sterility.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Infected/uninfected berry pericarps were ground into liquid nitrogen and total RNA was isolated from 200 mg of powders using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA). The RNA quantity, integrity and purity were controlled using a Nanodrop 2000 instrument (Thermo Scientific, Wilmington, DE, USA) and a Bioanalyzer Chip RNA 7500 serie II (Agilent).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the Low Input Quick Amp Labeling kit (Agilent Technologies) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop 2000 instrument (Thermo Scientific, Wilmington, DE, USA).
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| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent-048771 4x44K Grape all custom microarray chip (GPL22427) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to XDR Hi100%, XDR Lo 10%).
|
| Description |
Gene expression 5 days after the injection (Time 1) in water-injected berry pericarps, replicate 3
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5.1 (Agilent) using default parameters (protocol GE1-105_Dec08 and Grid:048771_D_F_20130410) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Jul 06, 2018 |
| Last update date |
Jul 26, 2019 |
| Contact name |
Teresa Colombo |
| Organization name |
Consiglio Nazionale delle Ricerche (CNR)
|
| Department |
Institute of Molecular Biology and Pathology (IBPM)
|
| Street address |
p.le A. Moro 7
|
| City |
Rome |
| ZIP/Postal code |
00185 |
| Country |
Italy |
| |
|
| Platform ID |
GPL22427 |
| Series (2) |
| GSE116740 |
Whole-transcriptome microarray analysis of grapevine-Botrytis cinerea interaction during latent infection of berries (“noble rot”) |
| GSE116741 |
Whole-transcriptome analysis of grapevine-Botrytis cinerea interaction during latent infection of berries |
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