Five microlit ers of Cy3 or Cy5 was added to each sample and incubated for 2 hrs in the dark at RT. Labeled aRNA was purified according to kit instructions and quantified using the Nanodrop spectropho tometer. One hundred pmol Cy3 and Cy5 labeled aRNA targets were denatured by incubating at 65 C for 5 min and added to a hybridization Vandetanib structure mix containing 9l 20 SSC, 5. 4l Liquid Block, and 3. 6l 2% SDS for a 90l total volume. Hybridization and data analysis Microarrays comprised of 70 mer oligonucleotides obtained from the University of Arizona were immobilized Inhibitors,Modulators,Libraries by rehydrating the slide over a 50 C waterbath for 10 s and snap drying on a 65 C heating block for 5 s for a total of four times. Slides were UV crosslinked at 180 mJ in a UV cross linker. The slides were then washed in 1% SDS, dipped in 100% EtOH five times followed by 3 min shaking.

Slides were spun dry at 1000 rpm for 2 minutes and immediately placed in a light proof box. The 90l hybridization mix was pipetted onto a microarray slide underneath a lifterslip and placed in a hybridization chamber overnight at 55 C. After hybridi zation, slides were washed in 2 SSC, 0. 5% SDS for 5 min utes at 55 C, 0. 5 SSC for 5 minutes at room temperature, and 0. 05 SSC for Inhibitors,Modulators,Libraries 5 minutes at room temperature. Slides were then spun dry at 1000 rpm in a Sorvall centrifuge and scanned with a GenePix 4000B scanner. The intensity variation was removed by fitting a loess regression using SAS 9. 1. Data were log 2 transformed and statistically analyzed using rank product statistics as described by to identify differentially expressed genes.

Bioconductor Rank Prod package was used to perform the rank product analysis. Significantly different genes reported in this study exhibited P 0. 001, as designated by the rank product analysis. The false discovery rate value obtained was based on 10,000 random permutations. Since Inhibitors,Modulators,Libraries 10,000 random permutations was very computer intensive, 1000 random permutations were performed 10 different times each time starting with a different random seed number and the average FDR value calculated was used for further analysis. The genes that had FDR values less than or equal to 0. 01 were considered as differentially expressed. Data for all microarray experiments were sub mitted to the NCBI GEO microarray database and can be viewed under the accession GSE10425.

Microarray Data Quality Control Global gene expression profiling comparing arsenate treated Arabidopsis plants with control was carried out to better understand the mechanisms of plant response to arsenate stress and to identify genes involved in arsenic metabolism. For microarray data quality Inhibitors,Modulators,Libraries control, we examined both dye dependent Inhibitors,Modulators,Libraries effects and distribution of the ratio after normalization. We have included an addi tional file that illustrates the quality of microarray experi ments, as well as the overall gene expression pattern. Additional file 2a shows the normal ized Gemcitabine structure M vs.