The aluminum (Al) treatment was initiated after 24 hours of growth, by replacing the nutrient solution with a solution of the same composition, also containing Al supplied as KAl(SO4)2 to a final free Al3+ activity of 39uM. Root samples were collected at 'time zero', and after 2, 6 and 24 hours of treatment
Growth protocol
Maize seedlings were germinated in wet filter paper for 3 days, then transferred to a full nutrient solution at pH 4.0 under aeration (Magnacava et al, 1987; Pineros et al, 2005).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIzol reagent following manufacturer's instructions. RNA was concentrated via NH4Ac/EtOH precipitation
Label
Cy3
Label protocol
10ug total RNA was reverse transcribed overnight at 42C using Superscript III (Invitrogen) in the presence of dNTPs and 5-(3-aminoallyl)-UTP. Samples were cleaned-up using Qiaquick PCR Purification kit (Qiagen) and dried on a speed vac. Amino-labeled cDNAs were coupled to Cy dyes using the CyDye Post-Labeling Reactive Dye Pack (Amersham Biosciences). cDNA pellets were resuspended in 0.1M Na2CO3 and allowed to couple to Cy dyes for 2 hours. Reactions were neutralized with 0.1M NaOAc, purified using Qiaquick PCR Purification kit and dried in a speed vac.
Hybridization protocol
Slides were pre-hybridized for 45 minutes at 42C in 5xSSC, 1%SDS, 1% BSA fraction V. Slides were then washed in water and isopropanol and spin-dried. Slides were hybridized overnight at 42C in 30% formamide, 5xSSC, 0.1%SDS, 5x Denhardts solution, and 0.1 ug/uL polyA RNA. Labeled cDNA was resuspended in 90uL of hybridization buffer and denatured at 95C for 2 minutes before being applied to the slide. Arrays were covered with Lifterslip coverslips (Erie Scientific) and placed in hybridization chambers (Corning). Afer hybridization, slides were washed sequentially once in 1xSSC 0.2%SDS, once in 0.1xSSC 0.2%SDS, and three times in 0.1xSSC. Slides were spin-dried and scanned immediately.
Scan protocol
Slides were scanned in a ScanArray Express scanner (PerkinElmer).
Description
Cohybridized with GSM260505
Hybridizations were performed technically as dual channel (eg, Cy3 and Cy5-labeled samples co-hybridized to the same array) but the results were processed as though they are single channel (Cy3 and Cy5 signals are treated independently; Cy3/Cy5 ratios are not calculated). See 'Data processing' below.
Data processing
LOWESS normalization was applied. The LOWESS-normalized signal intensity values were log2 transformed and analyzed by two interconnected ANOVA mixed models using PROC MIXED in SAS (Jin et al., 2001; Wolfinger et al., 2001). The normalization ANOVA model yij = µ + Ai + Dj + (A x D)ij + eij was applied to account for experiment-wide sources of variation associated with array (Ai, degrees of freedom (df) = 15, random effect), dye (Dj, df = 1, fixed effect), and their interactions. The residuals (eij) were treated as normalized values and analyzed in the following ANOVA model (gene model), where effects were evaluated for each gene individually: rikl = µ + Ai + Gk + Tl + (G x T)kl + ekl. Gk is the kth genotype (i.e. C100-6 or L53; df = 1) and represents the effect of each genotype on the expression of every gene, Tl represents the lth treatment (i.e. 0, 2, 6 or 24 hours of Al treatment; df = 3), and (G x T)kl the interaction between genotype and treatment (df = 3). Array (Ai) was included as a random effect to control for spot effects (Jin et al., 2001). Least square means were calculated, and estimates of differential expression were calculated as the difference between least-square means for each of the terms in the model.
