Seedlings with 2- to 3 cm root length were removed from agar plates and submerged in 1000 ml flasks containing 500 ml of modified one-sixth MS liquid medium containing 50 micromolar aluminum and 1% sucrose added as a carbon source for 16 hr.
100 seedlings were harvested at following 16 hr Al or mock treatment.Tissue was ground to a fine powder in liquid nitrogen using a pestle and mortar and total RNA was extracted using the following method. Each 0.5 to 1 g of powdered tissue were added to 5 cm3 of STAT-60 (Tel-Test, TX). The mixture was homogenized and stored on ice for 10 min. Chloroform/isoamyl (24:1, v:v) ETOH was added to the homogenate and centrifuged at 12000g for 30 min (40C). Following the transfer of the aqueous phase to a fresh tube, RNA was precipitated with isopropanol overnight at –200C, pelleted by centrifugation at 12000g for 30 min (40C), and resuspended 0.1 cm3 of RNase-free water. Total RNA was quantified using Beckman Spectrophotometer, and qualified by running 1 μg cm-1 of each sample onto a RNA Lab-On-A-Chip using a Agilent Bioanalyzer 2100 (Agilent technologies, Mountain View, CA).
Enzo Biotin-labeled in vitro transcribed (IVT)
Briefly, total RNA (8 mg) was synthesized to cDNA using the Superscript Double-Stranded cDNA synthesis kit (Invitrogen Corp, Carlsbad, CA) and poly dT-nucleotide primers that contain a sequence recognized by T7 RNA polymerase. The newly synthesized cDNA was used as a template to generate biotin-labeled invitro transcribed (IVT) cRNA using the Bio-Array High Yield RNA transcript labeling kit (Enzo Diagnostics, Inc, Farmingdale, NY).
Twenty micrograms of the cRNA was fragmented to strands of 35 to 200 bases in length. The fragment cRNA was hybridized to and Affymetrix ATH1 array GeneChip at 450C with rotation for 16 h (Affymetrix GeneChip Hybridization Oven 320). The GeneChip arrays were washed and stained (streptavidin phycoerythrin) on an Affymetrix Fluidics Station 400, followed by scanning.
Scanned using Affymetrix 3000 GeneChip scanner
The mRNA levels were measured using Affymetrix ATH1 chips (containing 22,810 probe sets) according to standard GeneChip Expression assay protocol. Total RNA was extracted from 100 pooled Arabidopsis seedlings for each GeneChip, control and Al-treated. A total of four chips were used in this experiment, representing two biological replicates to measure expression of whole plant material.
After hybridization, the chips were scanned using a GeneChip Scanner and raw hybridization data from microarray experiments were imported directly into Affymetrix GeneChip Operating software (GCOS) v1.2 for normalization and to determine the probe intensities and “Present” (P) or “Absent” (A) calls for each chip. The scanned image results for all 4 chips were quantified and analyzed using, dChip (Li and Wong, 2001) and RMA software (Irizarry et al., 2003). The gene list generated in dchip was uploaded into gene ontology consortium website (http://www.geneontology.org/) to further determine the functional categories of up- and down regulated gene expression.