|
| Status |
Public on Sep 15, 2013 |
| Title |
Tobacco-CMV-pepo-2 |
| Sample type |
RNA |
| |
|
| Source name |
Tobacco Leaf CMV-pepo-inoculation
|
| Organism |
Nicotiana tabacum |
| Characteristics |
disease state: Chlorotic tissue
tissue: leaf
treatment: CMV-pepo-inoculation
|
| Growth protocol |
The largest leaf of five leaf stages of tobacco (Nicotiana tabacum cv. Samsun) was mechanically inoculated with purified virus (100 μg/ml in phosphate buffer) or phosphate buffer only (mock control). For microarray analysis, four individual tobacco plants were used per each biological replication and three biological replications were prepared. The inoculated plants were grown in a growth chamber with a 16 h light (25 ˚C) / 8 h dark (22 ˚C) cycle.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from 100 mg leaf sample by RNeasy Plant Mini Kit with DNase I treatment (Qiagen) following the manufacturer's recommendations. RNA concentration and integrity were analyzed with an Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
For the linear T7-based amplification step, 100 ng of each total RNA sample was used. To produce Cy3-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies) following the manufacturer’s protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies).
|
| |
|
| Hybridization protocol |
1.65 μg Cy3-labeled fragmented cRNA in hybridization buffer was hybridized overnight (17 h, 65˚C) to Agilent Whole Tobacco Genome Oligo Microarrays 4x44K. Finally, the microarrays were washed once with the Agilent Gene Expression Wash Buffer 1 for 1 min at room temperature followed by a second wash with Agilent Gene Expression Wash Buffer 2 for 1 min at 37˚C. The last washing step was performed with acetonitrile.
|
| Scan protocol |
Fluorescence signals were detected using Agilent’s Microarray Scanner System (Agilent Technologies).
|
| Data processing |
The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files.
|
| |
|
| Submission date |
May 28, 2013 |
| Last update date |
Sep 16, 2013 |
| Contact name |
Tomofumi MOCHIZUKI |
| E-mail(s) |
tomochi@plant.osakafu-u.ac.jp
|
| Organization name |
Osaka Prefecture University
|
| Department |
Graduate School of Life and Environmental Sciences
|
| Street address |
1-1, Gakuen-cho, Naka-ku
|
| City |
Sakai |
| State/province |
Osaka |
| ZIP/Postal code |
599-8531 |
| Country |
Japan |
| |
|
| Platform ID |
GPL10098 |
| Series (1) |
| GSE47410 |
Quantitative transcriptional changes associated with chlorosis severity in mosaic leaves of tobacco plants infected with the Cucumber mosaic virus |
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