Seeds were sown on soil and leaves of 3-week-old seedlings were used for total RNA extraction
Growth protocol
Plants were grown on soil at 22°C under a 12-hr light/dark cycle and 70 % humidity
Extracted molecule
total RNA
Extraction protocol
RNeasy (Qiagen) according to the manufacturer protocol
Label
biotin
Label protocol
Biotinylated complementary RNAs (cRNAs) were in vitro transcribed from synthesized cDNA by T7 RNA polymerase (ENZO BioArray High Yield RNA Transcript Labeling Kit, Enzo). cRNAs were purified using affinity resin (Qiagen RNeasy Spin Columns) and randomly fragmented by incubating at 94 °C for 35 min in a buffer containing 40 mM Tris-acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate to produce molecules of approximately 35 to 200 bases long. (Zhu et al. (2001) Plant Physiol Biochem 39:222-242)
Hybridization protocol
The labeled samples were mixed with 0.1 mg/mLsonicated herring sperm DNA in a hybridization buffer containing 100 mM 2-N-morpholino-ethane-sulfonic acid (MES), 1 M NaCl, 20 mM EDTA, 0.01 % Tween 20, denatured at 99 °C for 5 min, and equilibrated at 45 °C for 5 min before hybridization. The hybridization mix was then transferred to the Arabidopsis GeneChip genome array (Affymetrix) cartridge and hybridized at 45 °C for 16 h on a rotisserie at 60 rpm. The hybridized arrays were then rinsed and stained in a fluidics station (Affymetrix). They were first rinsed with wash buffer A (6× SSPE (0.9 M NaCl, 0.06 M NaH2PO4, 0.006 M EDTA), 0.01 % Tween 20, 0.005 % Antifoam) at 25 °C for 10 min and incubated with wash buffer B (100 mM MES, 0.1 M NaCl, 0.01 % Tween 20) at 50 °C for 20 min, then stained with Streptavidin Phycoerythrin (SAPE) (100 mM MES, 1 M NaCl, 0.05 % Tween 20, 0.005 % Antifoam, 10 mg/mL SAPE, 2 mg/mL BSA) at 25 °C for 10 min, washed with wash buffer A at 25 °C for 20 min and stained with biotinylated anti-streptavidin antibody at 25 °C for 10 min. After staining, arrays were stained with SAPE at 25 °C for 10 min and washed with wash buffer A at 30 °C for 30 min. The probe arrays were scanned twice and the intensities were averaged with a Hewlett-Packard GeneArray Scanner. (Zhu et al. (2001) Plant Physiol Biochem 39:222-242)
Scan protocol
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner
Description
Gene expression data from an Arabidopsis line carrying silent HPT transgenes at A locus
Data processing
The data were analyzed with Microarray Suite version 4.0 (MAS 4.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100.
