|
| Status |
Public on May 18, 2013 |
| Title |
med14_12hr_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
med14_infected_12hr
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col-0
genotype/variation: med14 mutant
infected with: avirulent bacterial pathogen Pst DC3000/avrRpt2
time point: 12 hrs after infection
tissue: leaves
|
| Treatment protocol |
The bacterial pathogen Pst DC3000/avrRpt2 cell suspension (in 10 mM MgCl2, OD600 = 0.001) was infiltrated into Arabidopsis leaves with a 1 mL needleless syringe.
|
| 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 |
Total RNA was extracted from Arabidopsis leaves with a standard phenol-based method. RNA concentration was determined on a NanoDrop Spectrophotometer (Thermofisher Scientific, Waltham, MA) and sample quality was assessed using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| 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, 1650 ng of Cy 3 labeled cRNA was fragmented and hybridized with rotation at 65°C for 17 hr. Samples were hybridized to Arabidopsis 4X44k arrays (Agilent Technologies).
|
| Scan protocol |
The arrays were washed according to the manufacturer’s protocol and then scanned on a G2505B scanner (Agilent Technologies).
|
| Description |
SAMPLE 11
|
| Data processing |
Data were extracted using Feature Extraction 10.1.1.1 software (Agilent Technologies).
Data (individual signal intensity values) obtained from the microarray probes were background corrected using a normexp+offset method, in which a small positive offset (k = 50) was added to move the corrected intensities away from zero. The resulting data 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 value (R v.2.15.2). After normalization, the Student’s t-test was performed considering a probe-by-probe comparison between different genotypes at the same time point using wild type (Col-0) as the reference sample and between different time points of the same genotype using the 0-hr sample as the reference. In each comparison, a p-value and fold change (FC) were computed for each gene locus. The gene expression fold changes were computed based on the normalized log-transformed signal intensity data.
|
| |
|
| Submission date |
Mar 15, 2013 |
| Last update date |
May 18, 2013 |
| 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 (2) |
| GSE45212 |
Microarray analysis of med14 and wild type (Col-0) infected with the avirulent bacterial pathogen Pst DC3000/avrRpt2 |
| GSE45215 |
Microarray analyses of pathogen-induced transcriptome changes in the Arabidopsis mutant med14 |
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