cultivar: Col-0 tissue: leaves treatment: N starvation
Growth protocol
Arabidopsis seeds were germinated and pre-cultured on rockwool moistened with tap water for one week. Subsequently, plants were grown hydroponically under non-sterile conditions in a growth cabinet under the following conditions: 10 h/14 h light/dark; light intensity 280 μmol m−2 sec−1; day/night temperature 22°C/18°C and 60 % relative humidity. Tap water was substituted in the second week by half-strength nutrient solution containing 0.5 mM KH2PO4, 0.5 mM MgSO4, 125 μM K2SO4, 125 μM CaCl2, 50 μM Na–Fe–EDTA, 50 μM KCl, 30 μM H3BO3, 5 μM MnSO4, 1 μM ZnSO4, 1 μM CuSO4, and 0.7 μM NaMoO4, pH 5.8 with KOH. Nitrogen was supplied as 0.5 mM KNO3. In the third week, the plants were supplied with full-strength hydroponic growth medium (1 mM KH2PO4, 1 mM MgSO4, 250 μM K2SO4, 250 μM CaCl2, 100 μM Na–Fe–EDTA, 50 μM KCl, 30 μM H3BO3, 5 μM MnSO4, 1 μM ZnSO4, 1 μM CuSO4, and 0.7 μM NaMoO4, pH 5.8 with KOH), containing 1 mM KNO3. From week four until harvest (week nine), the plants were further cultivated in the full-strength growth medium, but nitrogen was supplied as 0.1 mM NH4NO3. The nutrient solution was renewed once a week during the first 3 weeks, twice in the fourth week and every 3 days for the following weeks.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from frozen and ground tissue according to Verwoerd et al. (1989. Nucleic Acids Res. 17, 2362) and purified with the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA was photometrically quantified with a NanoPhotometer (Implen, München, Germany) and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
Cy3
Label protocol
Cy3-labeled cRNA was synthesized with the Agilent Quick Amp Labeling Kit one-cCy3-labeled cRNA was synthesized with the Quick Amp Labeling Kit, one-color (Agilent Technologies catalog number 5190-0442) according to the manufacturer's instructions.
Hybridization protocol
1.5 ug of Cy3-labelled cRNA was fragmented in 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. Hybridization was performed in a Agilent hybridization oven at standard conditions as recommended by the manufacturer. Subsequently, microarrays were washed 1 minute with GE Wash Buffer 1 (Agilent) at 23°C and 1 minute with GE Wash buffer 2 (Agilent) at 37°C.
Scan protocol
Microarrays were scanned immediately after washing with an Agilent High-Resolution Scanner (G2505C) using default settings.
Description
Gene expression after 6 weeks N starvation
Data processing
The images were processed with the Agilent Feature Extraction software 10.1 using default settings (protocol GE1_105_Dec08 (Read Only) and GridName US91803681_252963410002_S01_grid (from grid_file)). The resulting processed signals of all individual hybridizations were quantile-normalized using the ranked median quantiles according to Bolstad et al. (2003. Bioinformatics 19, 185-193).
