|
| Status |
Public on Aug 19, 2011 |
| Title |
Roots_sclareol_2h_rep.3 |
| Sample type |
RNA |
| |
|
| Source name |
Roots, sclareol, 2h, replicate 3
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: root
|
| Treatment protocol |
Sclareol was purchased from (Sigma-Aldrish). Sclareol was dissolved in methanol and diluted to a final concentration of 100 uM with water. Roos of Arabidopsis plants grown on peat moss pellets were treated with 100 uM sclareol or with 0.1% methanol by immersing the roots into a solution containing the chemical and incubating for 2 h at 20C under continuous light.
|
| Growth protocol |
Sterilized seeds of Arabidopsis Columbia (Col-0) plants were placed on on half-strength Murashige and Skoog (MS) medium (Wako Pure Chemical, Osaka, Japan) supplemented with 0.7% agar and incubated for 7 days. The seedlings were transferred to peat moss pellets (Jiffy-7C, Jiffy Products of America Inc., OH) and grown under a cycle of 10 h of light and 14 h of dark at 20C for 3 to 4 weeks.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Roots samples in each treatment were harvested in triplicate and used for total RNA extraction. Total RNA was extracted using TRIzol reagent (Invitrogen) and purified using RNA purification columns (RNeasy, Qiagen) according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
500 ng RNA was labeled with cyanine-3 using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) and the labeled cRNA was purified using RNeasy mini spin columns (Qiagen) according to the manufacturer's instructions. Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
1.65 ug of Cy3-labeled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 1x Agilent fragmentation buffer and 2x 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 Oligo DNA microarray ver. 4.0 (catalog number G4136A) for 17 hours at 65C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 2 minute with 37C GE Wash Buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Each slide was scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5 um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after treatment with sclareol for 2 h
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) 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 |
Aug 05, 2011 |
| Last update date |
Aug 19, 2011 |
| Contact name |
Shigemi Seo |
| E-mail(s) |
sseo71@affrc.go.jp
|
| Phone |
+81-(0)29-838-7440
|
| Organization name |
National Institute of Agrobiological Sciences
|
| Department |
Plant and Microbial Research Unit, Division of Plant and Microbial Sciences
|
| Street address |
2-1-2 Kannondai
|
| City |
Tsukuba |
| State/province |
Ibaraki |
| ZIP/Postal code |
305-8602 |
| Country |
Japan |
| |
|
| Platform ID |
GPL9020 |
| Series (1) |
| GSE31230 |
Identification of natural diterpenes as inducers of resistance to bacterial wilt disease in tobacco, tomato, and Arabidopsis |
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