|
| Status |
Public on Jun 30, 2012 |
| Title |
Wild type_ 4hr |
| Sample type |
RNA |
| |
|
| Source name |
Wild type, infected, 4hr
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype/variation: Wild type
infection: avirulent bacterial pathogen Pst DC3000/avrRpt2
time point: 4 hr
tissue: leaf
|
| Treatment protocol |
Leaves of 4-week-old plats were inoculated with a 1 mL needless syringe by pressure-infiltration and leaf tissues were collected at the indicated time points.
|
| Growth protocol |
Arabidopsis seeds were sown on autoclaved soil (Metro-Mix 200, Grace-Sierra, Malpitas, CA) and vernalized at 4°C for 3 days. Plants were germinated and grown at 23 to 25°C under a 16-hr-light/8-hr-dark regime.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
About 100 mg leaf tissues were ground into a fine powder in liquid nitrgen and resuspended in 500 μL 80°C water-saturated phenol and 500 μL 80°C extraction buffer (100 mM LiCl, 100 mM Tris pH 8.0, 10 mM EDTA, 1% SDS). After brief vortexing and centrifugation at 14,000 rpm for 5 min, the aqueous phase was transferred to another cold tube containing 500 μL of 24:1 chloroform:isoamyl alcohol. After vortexing and centrifugation, the aqueous phase was transferred to a DEPC-treated tube with 1/10 volume of 3 M NaOAc and 2 volume of 100% ethanol. The tube was inverted to mix and placed at -80°C for 30 min and then spun at 14,000 rpm for 15 min. After removing the supernatant, the pellet was washed with 80% ethanol, dried briefly, and resuspended in DEPC-treated water.
|
| Label |
Cy3
|
| Label protocol |
cDNA was synthesized from 200 ng of total RNA and used as a template for in vitro transcription in the presence of T7 RNA Polymerase and cyanine labeled CTP’s using the Quick Amp Labeling kit (Agilent Technologies) according the manufacturer’s protocol. The amplified, labeled complementary RNA (cRNA) was purified using the RNeasy Mini kit (Qiagen, Valencia, CA).
|
| |
|
| Hybridization protocol |
For each array, 1.65 μg of Cy 3 labeled cRNA was fragmented and hybridized with rotation at 65°C for 17 hr. Samples were hybridized to Arabidopsis 4 × 44k arrays (Agilent Technologies). The arrays were washed according to the manufacturer’s protocol.
|
| Scan protocol |
The arrays were scanned on a G2505B scanner (Agilent Technologies).
|
| Description |
Gene expression 4 hr after Pst DC3000/avrRpt2 infection
|
| Data processing |
Data were extracted using Feature Extraction 10.1.1.1 software (Agilent Technologies).
Data (individual signal intensity values) obtained from the 8 microarray probes were log transformed (using 2 as the base) and normalized between individual samples by scaling the individual log-transformed signal intensities so that all datasets had comparable lower quartile, median and upper quartile values. Normalized, log2 signals generated using MATLAB.
|
| |
|
| Submission date |
Jun 28, 2012 |
| Last update date |
Jun 30, 2012 |
| Contact name |
Zhonglin Mou |
| Organization name |
University of Florida
|
| Department |
Microbiology and Cell Science & Plant Molecular and Cellular Biology
|
| Street address |
981 Museum Road
|
| City |
Gainesville |
| State/province |
FL |
| ZIP/Postal code |
32610 |
| Country |
USA |
| |
|
| Platform ID |
GPL12621 |
| Series (1) |
| GSE38999 |
Microarray analysis of At-med16-1 and wild type (Col-0) infected with the avirulent bacterial pathogen Pst DC3000/avrRpt2 |
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