strain/background: Col-0 genotype/variation: elp2 mutant tissue: leaves treatment: wounded, non-infected with Botrytis cinerea time point: 3 hr
Treatment protocol
Botrytis cinerea was grown on BD Difco Potato Dextrose Agar (Becton, Dickinson and Company, Sparks, MD) for about 10 days at 25°C. Spores were harvested, resuspended in BD Difco Potato Dextrose Broth (Becton, Dickinson and Company) at a density of 1-5×10^5 spores per mL, and incubated for 2 hr prior to inoculation. Five-μL spore suspensions were dropped on the adaxial surface of rosette leaves where the leaves were gently wounded with a needle. Leaf tissues were collected at the indicated time points. Leaves that were only gently wounded with a needle were also collected at 3 hr as controls.
Growth protocol
Arabidopsis seeds were sown on autoclaved soil (Sunshine MVP, Sun Gro Horticulture, Agawam, MA) 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 CTPs using the Quick Amp Labeling kit (Agilent Technologies) according to 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 Cy3 or Cy5 labeled cRNA was fragmented and hybridized with rotation at 65°C for 17 hr. Samples were hybridized to Agilent-021169 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 3 hr after wounding with a needle Replicate 2
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 values. The normalized data is generated by R(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
Jun 21, 2013
Last update date
Jul 01, 2013
Contact name
Zhonglin Mou
Organization name
University of Florida
Department
Microbiology and Cell Science & Plant Molecular and Cellular Biology
