|
| Status |
Public on May 22, 2009 |
| Title |
Mature_green_tomato_fruit_B. cinerea_Inoculated_biological_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
B. cinerea wound-inoculated fruit at mature green stage
|
| Organism |
Solanum lycopersicum |
| Characteristics |
Cultivar: Ailsa Craig
Fruit ripening stage: mature green stage (31 days post anthesis)
Treatment: wound-inoculated with B. cinerea (B05.10) conidia
|
| Treatment protocol |
B. cinerea strain B05.10 conidia were collected from sporulating cultures of the fungus grown on 1 % potato dextrose agar. Conidia concentration was determined using a Neubauer Ruling and then diluted to obtain a concentration of 500 spores/μL. On the day of harvest fruit were surface sterilized by submersion in a solution of 10% (v/v) bleach followed by three deionized water rinses. At the time of inoculation fruit were woundedto a depth of 2 mm and a diameter of 1 mm and inoculated with 10 μl of water suspension containing 5,000 conidia of B. cinerea. The wounding treatment was performed by wounding independent fruit as describe above followed by placing 10 μl of water at the sites of wounding. Fruit were incubated at 20°C in high humidity. Healthy and wounded fruit were kept exactly at the same conditions as the B. cinerea-inoculated fruit.
|
| Growth protocol |
Tomato plants (Solanum lycopersicum cv. Ailsa Craig) were grown in a field in the summer of 2008 in Davis, California. Standard cultivation and fertilization practices were used. Fruit were tagged 3 days after anthesis and harvested 31 days post-anthesis (dpa) as mature green and 42 dpa as red ripe.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA samples were extracted from fruit outer pericarp and epicarp tissues within a 0.5 cm diameter around and including the inoculation sites. Each biological replicate consisted of a pool of pericarp tissue collected around the inoculation sites (0.5 cm diameter) of at least 5 different fruit. Three grams of pericarp/epidermis tissue were ground in liquid nitrogen and 3 mL of a solution containing 100 mM Tris/HCl (pH 8.0), 25 mM EDTA, 75 mM NaCl, 1 % SDS and 1 M b-mecaptoethanol was added to the obtained fine powder. Three milliliters of phenol/chloroform/IAA (25/24/1, v/v/v, Tris/HCl 10 mM pH 7.9) were added to the mix and centrifuged 15 min at 10,000 g. Phenol/chlorophorm/IAA separation was repeated two or three times after collecting the supernatant obtained from each centrifugation. Nucleic acids were precipitated in 7.5 mL of ethanol and 300 μL of 3 M Na acetate pH 5.2 for 30 min at -20 °C. The pellet obtained by centrifuging 15 min at 12,000 g was resuspended in 1 mL of water and once completely solubilized 250 μL of 8 M LiCl solution were added. After overnight precipitation and centrifugation for 15 min at 12,000 g at 4 °C, the pellet was washed with 70% ethanol and the resuspended nucleic acids were treated with DNase (RQ1 RNase-Free DNase, Promega) using the manufacturer's recommended protocol. The RNA solution was subsequently washed once in phenol/chloroform/IAA (25/24/1, v/v/v, Tris/HCl 10 mM pH 7.9) and twice in chloroform/IAA (24/1, v/v). Two microliters of the isolated RNA were used to estimate nucleic acid concentration and purity with the Thermo Scientific NanoDropTM 1000 Spectrophotometer. An aliquot of the total RNA was checked for integrity by running on an agarose gel.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 10 μg total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
|
| |
|
| Hybridization protocol |
Using the manufacturer's recommended protocols, the arrays were washed, stained, and scanned by the Microarray Core Facility (UC Davis School of Medicine).
|
| Scan protocol |
The biotinylated cRNA was fragmented and then hybridized to the GeneChip® Tomato Genome Array. Using the manufacturer's recommended protocols, the Tomato arrays were washed, stained, and scanned by the Microarray Core Facility (UC Davis School of Medicine).
|
| Description |
Gene expression data from B. cinerea (B05.10) wound-inoculated mature green tomato fruit
|
| Data processing |
The raw hybridization data were imported into the R software environment (R Foundation for Statistical Computing, Wien; http://www.R-project.org) and the R/affy package from Bioconductor (http://www.bioconductor.org) was used to preprocess the data. The raw data were background corrected, normalized and summarized using the robust multi-array average (RMA) expression measure).
|
| |
|
| Submission date |
Jan 29, 2009 |
| Last update date |
May 22, 2009 |
| Contact name |
Dario Cantu |
| E-mail(s) |
dacantu@ucdavis.edu
|
| Organization name |
UC Davis
|
| Department |
Plant Sciences
|
| Street address |
One Shields Ave
|
| City |
Davis |
| State/province |
CA |
| ZIP/Postal code |
95616 |
| Country |
USA |
| |
|
| Platform ID |
GPL4741 |
| Series (1) |
| GSE14637 |
Expression data of tomato fruit responses to Botrytis cinerea |
|
