|
| Status |
Public on Jan 30, 2015 |
| Title |
Nobeokabouzu-komugi 3 DAI treated_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Nobeokabouzu-komugi florets_treated_3 days after inoculation
|
| Organism |
Triticum aestivum |
| Characteristics |
genotype: Nobeokabouzu-komugi
infected with: F. graminearum suspension
tissue: floret
time point: 3 day after inoculation
|
| Treatment protocol |
At the early anthesis time, single floret inoculation with F. graminearum strain H3 was carried out by pipetting 10μl of the fungal suspension (1x10^5 macroconidia ml-1) between lemma and palea of each floret. Same time control plants (mock) were inoculated with 10 μl of distilled water. The inoculated spikes were covered with a plastic bag for 72 hours.
|
| Growth protocol |
Plants were grown in glass without any stress, the temperature and moisture inside the glass house were maintained 25 °C and 50%respectively
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the florets by using Nucleo Spin RNA plant kit (Macherey-Nagel, Germany)
|
| Label |
Cy3
|
| Label protocol |
Total RNA was converted to cRNA labeled using Low Input Quick Amp Labeling kit (Agilent technologies) and fluorophore cy3-CTP. The samples were cleaned using RNeasy MinElute Clean up kit (Qiagen).
|
| |
|
| Hybridization protocol |
600ng of Cy3-labelled cRNA (specific activity >6 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25ml containing 25x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55μl of 2x GE Agilent hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent-015520 Custom wheat 38k array for 17 hours at 65°C in a 10rpm 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
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
NT3-1
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5.1.1 (Agilent) using default parameters (protocol GE1-v5_95 and Grid: 015520_D_F_20061122) 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 24, 2014 |
| Last update date |
Jan 30, 2015 |
| Contact name |
Alagu Manickavelu |
| E-mail(s) |
manicks@yokohama-cu.ac.jp
|
| Phone |
+81458202404
|
| Organization name |
Yokohama City University
|
| Lab |
Plant Genetics Resource Division
|
| Street address |
Maioka 641-12
|
| City |
Yokohama |
| State/province |
Kanagawa |
| ZIP/Postal code |
244-0813 |
| Country |
Japan |
| |
|
| Platform ID |
GPL9805 |
| Series (1) |
| GSE59721 |
Altered Gene Expression Profiles of Wheat Genotypes against Fusarium Head Blight |
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