finally, we ensured that known markers for specific types of neurons, allowing identification of individual cell types, were present on the chip. we paid special attention to genes associated with the differentiation and maturation of dopaminergic neurons. in many published studies, the expression of only a single ( tyrosine hydroxylase, th ) or 2 – 3 markers for dopaminergic neurons ( e. g. amino acid decarboxylase ( aadc ), dopamine transporter ( dat ), vesicular monoamine transporter 2 ( vmat2 ) ) have been used to indicate dopaminergic identity of neurons. in contrast, the neurostem chip includes oligo nucleot ide probes for 88 genes related to dopaminergic neurons, thus being more comprehensive in this sense, compared to other existing microarray platforms, including focused ones [ 17, 18 ]. those entries encompass recognized and candidate markers for dopaminergic neurons ( mature and early ) and progenitors, as well as markers for the maturation and differentiation of the latter ( table 1 ). table 2 represents conditional functional breakdown of genes targeted by the neurostem microarray platform. a number of important gene groups that are included in the chip are not mentioned in table 2. among these, entries related to dickkopf gene family, galanin -, melatonin -, vasoactive intestinal peptide ( vip ) -, camp response element - binding protein ( creb ) - and b cell leukemia 2 ( bcl2 ) oncogene - related are present. many of them play potentially important, yet undefined, roles in the biology of stem cells. additionally, we included some genes implicated in disease mechanisms of neurodegenerative disor ders ( most importantly, parkinson ' s disease and alzheimer ' s disease ) in the chip. furthermore, we incorporated a number of markers for distinc t differentiation pathways ( e. g. hematopoietic and pancreatic ) and cell types ( e. g. cancer subtypes and a range of normal cell types ) to serve as essential controls. taken together, we believe that in its present form neurostem chip represents currently most comprehensive gene expression platform for studies on stem cells, neural / neuronal differentiation, human neurodegeneration and neuronal survival, both in vivo and in vitro. the complete layout of neurostem chip will be disclosed to the academic community, upon request. the microarray format we selected relies on long oligo nucleot ide molecules ( 69 – 71 nucleot ides ) printed over a solid surface. we spotted the synthesized oligo nucleot ides ( operon biotechnologies ) with a constant concentration across the slides, and evaluated the quality and consistency of spotting in a series of control experiments. we then illustrated the utility and technical reliability of the neurostem chip by characterizing the gene expression profile of commonly utilized hesc line sa02 ( sahlgrenska 2; [ 27 ] ), including ( i ) undifferentiated cells and ( ii ) cells committed towards neuronal / dopaminergic differentiation pathway. for the first of these, we used total rna sample purified from hesc colonies that exhibited morphology consistent with cell proliferation and the absence of spontaneous differentiation ( figure 1a ). we also evaluated the expression of the cell cycle marker ki67 and the pluripotency marker oct3 / 4 in the sample by immunocytochemistry ( figure 1b – e ). co - culturing of escs with murine stromal cells ( including pa6 cell line ) rapidly generates dopaminergic neurons from escs by an unexplained mechanism termed stromal cell - derived inducing activity ( sdia; [ 28, 29 ] ). we therefore committed hescs toward the neuronal / dopaminergic differentiation pathway by co - culturing with pa6 cells for 16 days, resulting in appearance of cells positive for early and late neuronal markers, including nestin, β - iii - tubulin, and th, the established marker of dopaminergic neurons ( figure 2 ). to verify the expression of some key stem cell - and neural phenotype - associated genes we performed rt - pcr comparing rna samples from the undifferentiated hescs with hescs of the same line differentiated toward neuronal / dopaminergic pathway, as described above. the expression profile outlined by rt - pcr confirmed the identity of the sample used ( figure 3 ). after performing rna integrity tests, we incorporated fluorescent labels to the amplified rna samples from hescs ( cyanine 3 - ctp ( cy3 ) and cyanine 5 - ctp ( cy5 ) ), hesc - derived cells containing th - positive neurons ( cy3 and cy5 ) and human universal reference rna ( cy5 ), and hybri diz ed aliquots with neurostem microarray slides using the following conditions: hesc vs. reference, cy3: cy5 = ( i ) 20: 10 pmol, and ( ii ) 10: 5 pmol; and hesc vs. hesc - derived cells, cy3: cy5 = ( iii ) 30: 20 pmol, respectively. universal reference rna has been previously established as a standard reference material for microarray experiments, proving an ability to effectively hybri diz e to a large fraction of microarray spots [ 30 ]. we performed two - color hybri diz ations ( e. g. for the experiment vs. reference ) following an established protocol [ 31 ], and included dye - flip technical replicates in the analysis ( figure 4 ). using the online software program base [ 32 ] we sequentially filtered the data by background subtraction, negative flagging, negative intensities and for inconsistent data amongst replicates [ 33 ]. figure 5a shows a comparison of the spot intensities prior to normalization ( m versus a plot ), with the log2 of the expression ratio between cy3 / cy5 being plotted as a function of the log10 of the mean of the total expression intensities for cy3 and cy5 channels. the deviation of the line from zero revealed a need for normalization, so prior to data analyses we normalized signals using a locally weighted scatterplot - smoothing regression ( lowess ) algorithm ( figure 5a – b; fitted line ) implemented in base. since the reproducibility of two - color microarray gene expression data is critically important, we calculated pearson correlation coefficients of the reporters present in the filtered database comparing the average expression ratios ( 7005 for hescs vs. universal reference; 6947 for undifferentiated vs. neuronal / dopaminergic lineage - committed hescs ). results obtained revealed that data were consistent across technical replicates ( dye - swap and amount of loaded material ), showing general high reproducibility: e. g., correlation coefficients were greater than 0. 96 for technical replicates and 0. 78 for dye - swapping samples in hescs vs. universal reference hybri diz ations ( table 3 ). to detect genes with high expression levels in hesc samples, we filtered data for intensity values > 100 in the hesc sample and performed clustering analysis using the tigr multiexperiment viewer ( mev; [ 34 ] ). to visualize variations of spot / reporter per technical replicate, hierarchical clustering was performed by k - means classifier based on the linear - correlation - based distance ( pearson, centred ) method. the optimal number of clusters was determined empirically to produce the most balanced ratio of entries to cluster of highly expressed genes. a cluster of 101 genes up - regulated in the hesc sample [ see additional file 1 ], was plotted in a centroid graph ( figure 5c ); the variation across technical replicates was low. we merged technical replicates to generate a list of the most up - regulated genes expressed in the hesc sample compared to the universal reference rna ( table 4 ). standard error of the mean expressed as percentage was calculated for the 4 technical replicates, and was 6. 7 % for the top 25 genes up - regulated in hesc samples, compared to universal reference rna. we performed the analysis of microarray data, as described in the methods, and spot error values were generally in the lower range, indicating high stringency of the signals and low variance.