and set III, cDNA samples of salt treated ABR17 and untreated ABR17 transgenic seedlings for selleck chemicals llc hybridization to the oligo nucleotide arrays. Each microarray experiment consisted of six hybridizations according to the principles of dye swap design on tissues across three biological repli cates of the experiments. RNA was isolated using the QIAGEN RNeasy Plant Mini Kit from 2 week old WT and ABR17 seedling tissue from all three set of experiments and the integrity of all RNA samples assessed by agarose gel electrophoresis. For microarray hybridization, 6g of total RNA was used to synthesize cDNAs using SuperScript II RT with RT polyA capture primers in 3D Array 900TM. In these microarray experiments, 70 mer oligonucleotide arrays were used which contained 26,090 probes, plus additional probes for quality control.
Oligonucleotide arrays were spotted on superamine aminosilane coated slides. Each pair of sam ples within each of the three biological replicates was labeled in a reciprocal dye swap design, for a total of 18 hybridizations in all three sets of experiments. Slides Inhibitors,Modulators,Libraries were scanned using ArrayWoRxe and spot intensi ties were measured, quantified, normalized and analyzed using TM4. Spots with intensity ratios that differed significantly from 0 were identified by Stu dents t Inhibitors,Modulators,Libraries test. This procedure highlights the spots that dem onstrated statistically significant differential expression between the different samples. The raw microarray data of 18 hybridizations as well as the protocols used to produce the data were deposited in the ArrayExpress database.
Quantitative real time PCR validation of microarray data Primers for qRT PCR were designed using the Primer Express software to ensure that PCR products Inhibitors,Modulators,Libraries of approximately 70 80 bp were generated. cDNA syn thesis and qRT PCR analysis of gene expression of 19 genes were performed using the Taqman system as described previously on an ABI Prism 7700 Sequence detector and the SNP RT template program or using the SYBR green system as described by Yang and others was used to validate the expression of 8 genes. In both cases, the delta delta method was used to calculate relative gene expression using actin as the endogenous control. The rel ative transcript abundance in the controls was normalized to 1 and was used as a basis for comparison to the treat ments.
Plant Inhibitors,Modulators,Libraries tissue from three biological replicates was used in qRT PCR experiments and reactions for each bio logical replicate were performed in duplicate. Background Plants are well known for their extraordinary capacity to regenerate whole organisms from somatic cells. They often retain plasticity and have the capability to reverse the differentiation process and change their Inhibitors,Modulators,Libraries fate. The remarkable plasticity of plant cells is well exemplified by the capability selleck chem of differentiated leaf cells to retain totipo tency, the ability of a single cell to develop into a new organism.