Cells from all 12 donors were treated with each experimental condition. The microarray data collection was in compliance with the Minimum Information About Microarray Experi ments standard. The quality of the RNA was checked by the Agilent Bioanalyzer, and the quality of the hybridiza tion image was checked by the affyPLM model. To deal with the technical variation, each gene was measured by 11 except different probes on the Affymetrix U133A microar ray. A statistical model at the probe level was used to identify the differentially expressed genes. To estimate the variance more efficiently with a small sample size, we utilized an empirical Bayesian correction of the linear model. Statistical significance was considered with a P value of P 0. 001 and fold change larger than 1.

5 fold between the treatment group and corresponding control. All the data analysis was conducted using the Bioconduc tor R package. To interpret the biological signifi cance of differentially expressed genes, a gene ontology analysis was conducted Inhibitors,Modulators,Libraries using DAVID EASE. Pathway analysis and classification by gene ontology Regulated genes were used as input for both analyses. The ingenuity pathway analysis system was used to project genes onto known biolo gical pathways. The system deter mines a significance value for each pathway based on an F statistics that the input genes occur randomly within this pathway. Grouping of genes was done by computing over representation of regulated genes in gene ontology classes.

Statistical analysis consisted of 1 ana lysis Inhibitors,Modulators,Libraries of differentially expressed genes under a single experimental condition in comparison to the correspond Inhibitors,Modulators,Libraries ing control, 2 analysis of differentially expressed genes when comparison is made between two treatments, and 3 gene ontology, Inhibitors,Modulators,Libraries when changes were analyzed within a family of genes according to their function. Selected gene array results were verified experimentally in vitro or by real time PCR. Validation experiments quantitative real time PCR Selected gene array results were verified by real time PCR. SuperScript III reverse transcriptase with oligo 12 18 was used to transcribe 4 ug of isolated total RNA into complementary DNA in a total volume of 20 ul according to the manufacturers instructions. Real time PCR primer sets spe cific for human b actin, GAPDH, gremlin 1, IL 6, IL 8, and LIF 1 were designed using the Primer Quest program.

The specificity of the primers was verified by testing in BLAST searches. Real time PCR primer sets specific for human 18SrRNA and BMP 2 were purchased from Qiagen. Real time PCR was performed using the Smart Cycler System. Each 50 ul reaction mixture con tained 1X Platinum Quantitative PCR SuperMix UDG, 0. 5X Smart Cycler additive reagent, 0. 5X SYBR Inhibitors,Modulators,Libraries Green 1, 0. 2 uM each of forward selleck chem and reverse primer or 1X QuantiTect primers and 1 ul cDNA or 2 ul cDNA.