genotype: antisense-ACLA mutant treatment: harvested at 0.5h in the dark in the diurnal cycle under a SD photoperiod (8 h light/16 h dark). tissue: rosette leaves
Treatment protocol
Leaf samples were frozen in liquid N2 immediately after harvest and stored at -80‚°C for RNA extraction. The experiment was done twice and independent randomizations for plant growth and harvest were used for the two replicates.
Growth protocol
The antisense-ACLA mutant and WT plants were arranged according to a Randomized Complete Block Design. The plants were planted in rows with seven rows in each flat; two plants of the same genotype/pot. Plants were grown under a SD photoperiod (8 h light/16 h dark) in a growth chamber as described. Eight randomly selected rows were harvested for each time point from different flats. Plant material was harvested at 11 time points in the diurnal cycle (0, 0.5,1, 4, 6, 8,12 h in the dark condition, and 0, 0.5, 1, 4 h in the light condition; Time 0 is the beginning of the light period); harvesting was conducted under a green safety light. Each sample consisted of rosette leaves (leaves 5 to 8, staged following Bowmann (1994); photosynthetically active (Stessman et al., 2002)) from sixteen six-week-old plants.
Extracted molecule
total RNA
Extraction protocol
Leaf samples were frozen in liquid N2 immediately after harvest and stored at -80‚°C for RNA extraction through the standard Trizol protocol.
Label
biotin
Label protocol
Synthesis of labeled cRNAs, hybridization with Affymetrix Arabidopsis ATH1 arrays (22,810 Arabidopsis probe sets, Affymetrix, Santa Clara, CA),
Hybridization protocol
Synthesis of labeled cRNAs, hybridization with Affymetrix Arabidopsis ATH1 arrays (22,810 Arabidopsis probe sets, Affymetrix, Santa Clara, CA),
Scan protocol
scanning of the probe arrays were performed at the Iowa State University Gene Chip Facility (Ames, IA). Hybridization intensities were collected by an Agilent GeneArray Scanner (Agilent Technologies, Palo Alto, CA).
Description
Replicate2: antisense-ACLA material was harvested at 0.5h in the dark in the diurnal cycle under a SD photoperiod (8 h light/16 h dark).
Data processing
Raw data were processed with the Exon Array Computational Tool (ExACT) (Affymetrix) for background correction and normalization. Data analysis and statistical evaluations were performed with customized R codes (version 2.3.1, http://www.r-project.org/). We defined a probeset as present when it had a P value <0.001 and an Intensity value >200. These criteria were suggested by the results of preliminary experiments. Algorithm: ExpressionStat 5.0
