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RNA preparations were checked for RNA integrity by Agilent 2100 Bioanalyzer. All samples in this study showed high quality RNA Integrity Numbers (RIN) of 10. RNA was quantified by photometric Nanodrop measurement. Synthesis of cDNA and subsequent biotin labelling of cRNA was performed according to the manufacturers' protocol (3? IVT Express Kit; Affymetrix, Inc.). Briefly, 100?ng of total RNA was converted to cDNA, Crenolanib followed by in vitro transcription and biotin labelling of aRNA. After fragmentation labelled aRNA was hybridized to Affymetrix PrimeViewTM Human Gene Expression Microarrays for 16?h at 45?��C, stained by streptavidin/phycoerythrin conjugate and scanned as described in the manufacturer's protocol. Data analyses on digitized fluorescence signal intensities were conducted with GeneSpring GX software (Vers. 12.1; Agilent Technologies). Probes within each probeset were summarized by RMA after quantile normalization of probe level signal intensities across all samples to reduce inter-array variability (Bolstad et al., 2003). Input data pre-processing was concluded by baseline transformation to the median of all samples. After grouping of samples according to their respective experimental condition (USSC d0, USSC 16?h and USSC 5?d, three replicates each) a given probeset had to be expressed above background (i.e. fluorescence signal of a given probeset was detected within the 20th and 100th percentiles of the raw signal distribution of a given array) in at least 66% of replicates in any one of three groups to be further analysed. Differential gene Antidiabetic Compound Library screening expression was statistically determined by ANOVA analysis (p?RecBCD containing 278 differentially expressed probeset IDs were subjected to the ��Functional Annotation Cluster Tool�� provided by DAVID Bioinformatics Resources (http://www. david.abcc.ncifcrf.gov/) (Dennis et al., 2003, Huang da et al., 2009?and?Sherman et al., 2007). In a first approach, it was hypothesized that HOX negative USSCs might have the potential to adapt the HOX code from surrounding cells, as it was described in mice by Leucht et al. for mandibular periosteal cells (HOX?), expressing HOX genes after co-culture with tibial periosteal cells (HOX+) ( Leucht et al., 2008). CDSCs display a neonatal subpopulation differing from USSCs by their positive HOX code. Therefore, we decided to use this neonatal model system as it is likely that these celltypes come in close contact in vivo. A transwell system was applied to test this hypothesis. USSCs were co-cultured for 5?days with a CDSC line in the transwell insert. Afterwards HOX gene expression in USSCs was tested by conventional reverse transcriptase PCR (RT-PCR). In none of the experiments performed, USSCs developed a positive HOX expression pattern (data not shown here).