normalization data transformation protocol
Data was processed using GeneSpring GX10 Expression software (Agilent Technologies) and global scaling as normalisation method. Differentially expressed genes were identified using a two-class t-test (p< 0.05 significance level). Genes that were up or down-regulated more than 2-fold were selected ID_REF = VALUE = genespring normalized
array scanning protocol
GeneChips were scanned using an Affyimetrix GCS3000 scanner with Command Console Software (AGCC)
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Arabidopsis ATH1 array.
The cDNA was labelled using a MessageAmp(tm)II-Biotin Enhanced Kit (Ambion)
nucleic acid extraction protocol
Total RNA was extracted usign Qiagen RNAeasy kit for plants. RNA quality was tested using a Bioanalyser (Agilent Technologies)
For the microarray experiment, Arabidopsis was grown according to the method described by Kumari et al. (Kumari et al. 2008). Rafts were made from circular lightweight plastic, 75 mm diameter and 6 mm thick. Approximately 100 holes (3-4 mm diameter) were drilled into each disk. These rafts were sterilised by autoclaving and the holes were plugged with 1/2MS(A). Sterile Arabidopsis seeds, which had been stratified for two nights in the dark at 4 degrees C, were pipetted onto each plugged hole. Rafts were transferred to liquid Richard's medium (pH 5.7). Plants were grown in sealed sterile jars for 14 days at 22 degrees C / 19 degrees C day / night temperatures on a 16 hour light (80 umol.m-2.s-1) / 8 hour dark cycle.
sample treatment protocol
0.125 mM KAuCl4 was prepared in water and pH adjusted to pH 5.7 using NaOH. 1/2 Murashige and Skoog (1962) liquid growth medium was replaced with 0.125 mM KAuCl4 solution for six hours, and plants grown at 80 umol.m-2.s-1 21 oC for 6 hours. then roots harvested, snap frozen in liquid nitrogen and total RNA extracted.