xperiments Nutlin 3a All procedures were done as described in. Custom designed, full genome Arabidopsis oligonucleotide micro arrays printed at the Norwegian Microarray Consortium were used in all experiments. Quantitative real time PCR Inhibitors,Modulators,Libraries For qRT PCR analysis, the total RNA was DNAse trea ted using DNA free Kit, while the QuantiTect kit was used for cDNA synthesis. A LightCycler 480 System and the corre sponding SYBR Green I Master mix were used in a three step programme including preincubation at 95 C for 5 min, 40 cycles of amplification consisting of 95 C for 10 s, 55 C or 60 C for 10 s and 72 C for 10 s, and melting curve analy sis by heating from 65 C to 97 C with a ramp rate of 2. 2 C s. Each 20 ul reaction contained 0. 5 uM of each forward and reverse primer, and cDNA quantity Inhibitors,Modulators,Libraries corresponding to 50 ng of RNA.

LinRegPCR software was used to determine the PCR reaction Inhibitors,Modulators,Libraries efficiency for each sample and the effi ciencies for each primer set were calculated by averaging the efficiency values obtained from the individual sam ples. Relative expression ratios of the targeted genes were calculated and normalized to TIP41 like gene with the use of REST 2008 software. The qRT PCR analysis was performed with the use of three biological replicates. Statistical analysis of microarray data The microarray experiment was a 2 by 3 factorial, with the factors as plant type and treatment. Each experimen tal condition, i. e. each combination of factors, was repre sented by four biological Inhibitors,Modulators,Libraries replicates. Seven different direct comparisons of the experimental conditions, using four replicates for each comparison, were made with the use of microarray data sets.

However, only data from microarrays with very good technical quality were used for further analyses. Note that using this setup means Drug_discovery that the same biological replicate will occur on two different microarrays. Also note that experimental condi tions that were not compared directly can still be con trasted, but with lower efficiency than the direct comparisons. The microarray data for each array were filtered and normalized as discussed in. To make statistical infer ences about differential regulation between experimental conditions, the limma package was used. In each comparison of experimental conditions a q value was calculated for each gene. For a gene to be considered dif ferentially regulated at a statistically significant level, its q value had to be lower than 0.

05. In effect this controlled the false discovery rate of the comparison at a 0. 05 level. Aphid fitness experiments B. brassicae fitness http://www.selleckchem.com/products/BIBW2992.html on aos and fou2 mutants in compari son to wt Col 0 was evaluated in experiments assessing aphid asexual fecundity. Two first instar nymphs were placed on each plant and plants were placed in plexi glass cages. Eleven cages were used for each genotype tested. After 13 days, aphid progeny numbers in each cage were counted. To com pare aphid counts for the different plant types, a two tailed Wilcoxon rank sum test was used with