gene - level signal estimates suggested that notch was significantly higher in hcns - scns relative to hescs, but levels of notch were not significantly different in the derived nps compared to hescs. delta / notch - like egf - related receptor ( dner ), a neuron - specific transmembrane protein, was recently shown to bind to notch at cell – cell contacts and activates notch signaling in vitro [ 59 ]. rt - pcr validation of dner confirmed array - derived signal estimates, indicating an enrichment of dner in nps relative to hescs ( figure 1c ). finally, sox1, a hmg - box protein related to sry, was shown to be one of the earliest transcription factors expressed in cells committed to the neural fate [ 60 ]. here the gene - level estimates indicated that sox1 was expressed significantly higher in nps relative to hescs ( p - value 0. 00013, figure 1b ), a finding that was confirmed by rt - pcr ( figure 1c ). from these examples, we concluded that rt - pcr validation correlated well with gene - level estimates from the exon array. in addition, the derived nps had decreased levels of pluripotent markers oct4 and nanog but had levels of sox1 that were comparable to hcns - scns. this finding confirmed that the derived nps were committed to a neural fate and validated the use of hcns - scns as a benchmark for nps. next we asked whether the highest enriched genes in hescs relative to nps reflected our existing knowledge in the literature. using the above - mentioned groupings of hescs ( cyt - es, hues6 - es ) and nps ( cyt - np, hues6 - np, and hcns - scns ), 2, 945 genes were enriched in hescs relative to nps; and 552 genes were enriched in the nps relative to hescs, at a p - value significance cutoff of 0. 01 ( correcting for multiple hypothesis testing using the benjamini - hochberg method ). the 15 most enriched genes in hescs included genes such as teratocarcinoma - derived growth factor 1 ( tdgf1 / cripto; p - value < 10−12 ), zinc finger protein 42 ( zfp42 / rex1; p - value < 10−12 ), oct4 ( p - value < 10−12 ), nanog ( p - value < 10−10 ), lin - 28 homolog ( p - value < 10−10 ), cadherin 1 preprotein ( p - value < 10−10 ), claudin 6 ( p - value < 10−9 ), ephrin receptor epha1 ( p - value < 10−9 ), and erbb3 ( p - value < 10−9 ). tdgf1 / cripto was first shown to stimulate dna synthesis and cell proliferation of both undifferentiated and differentiated embryonic carcinoma cells [ 61 ] and was later shown to be important for cardio myoc yte formation from mouse esc [ 62 ]. oct4, reviewed in [ 63 ], and nanog [ 64 ] are crucial for the pluripotency of hescs. recently, knockdown of zfp42 / rex - 1 in mouse esc caused the cells to differentiate [ 65 ]. our gene - level exon array analysis confirmed that the hescs and nps were molecularly distinc t. to reveal global functional differences between the enriched genes in hescs or nps, the enriched genes were subjected to a gene ontology ( go, http: / / www. geneontology. org ) analysis as described previously [ 55 ]. enriched genes in hescs were more likely to be in molecular function categories, such as “ rna binding ” ( p - value < 10−12 ), “ structural constituent of ribosome ” ( p - value < 10−51 ), “ exonuclease activity ” ( p - value < 10−6 ), “ cytochrome - c oxidase activity ” ( p - value < 10−5 ), and “ atp binding ” ( p - value < 10−6 ), and in biological processes involved with “ trna processing ” ( p - value < 10−6 ) and “ protein biosynthesis ” ( p - value < 10−48 ), consistent with our knowledge of hescs as a rapidly proliferating population of cells ( figure 2a ). similar analysis of the enriched genes in nps revealed an overrepresentation in molecular functional categories, such as “ calcium ion binding ” ( p - value < 10−8 ) and “ structural molecule activity ” ( p - value < 10−5 ), and in biological processes involved with “ neurogenesis ” ( p - value < 10−38 ), “ cell adhesion ” ( p - value < 10−13 ), “ cell motility ” ( p - value < 10−4 ), “ development ” ( p - value < 10−6 ), “ neuropeptide signaling pathway ” ( p - value < 10−4 ), and “ endocyt osis ” ( p - value < 10−4 ) ( figure 2b ). considering that these were the only categories that were significantly enriched out of more than 18, 000 go terms, and that randomly selected sets of similar numbers of genes did not reveal statistical differences in go categories, our results confirmed that the global molecular profiles derived from exon array analysis were consistent with known differences between hescs and nps. figure 2gene ontology analysisdifferential gene expression of hescs ( cyt - es and hues6 - es ) and nps ( cyt - np, hues6 - np, and hcns - scns ) was computed from gene - level signal estimates. statistical significance for differential gene expression was determined by using t - statistics with benjamini - hochberg correction for false discovery rate ( p < 0. 01 ). gene ontology “ molecular function, ” “ cellular component, ” and “ biological process ” categories, which differed significantly ( p < 0. 05 ) in the representation between significantly enriched genes ( black bars ) and all other genes ( white bars ), were shown. statistical significance for go analysis was assessed by using χ2 statistics with bonferroni correction for multiple hypothesis testing. go categories are ordered from top to bottom in order of decreasingly significant bias toward enriched genes. ( a ) go analysis of enriched genes in hescs. ( b ) go analysis of enriched genes in nps. to summarize, firstly immunoh istochemical and rt - pcr evidence validated that the cells exhibited expected characteristics; secondly, stage - specific marker gene differences by rt - pcr were reflected ac curate ly by gene - level estimates from the exon arrays; thirdly, the hesc - enriched genes were coherent with known genes that controlled pluripotency and self - renewal; and lastly, the global functional profiles exemplified expected biological differences between hesc and np cells. description of the regression - based exon array protocolconvinced that the signal estimates from the exon arrays reflected expected molecular and biological differences between hescs and nps, we sought to identify as events. we compared cyt - es to hcns - scns to illustrate our approach. first we normalized the data and generated signal estimates with robust multichip analysis ( rma ) and estimated the probability that each probeset was detected above background ( dabg ) using publicly available affymetrix power tools ( apt ). we analyzed probesets that ( i ) comprised three or more individual probes; ( ii ) were localized within the exons of our gene models with evidence from at least three sources ( mrna, est, or full - length cdna ); and ( iii ) were detected above background in at least one of the cell lines. in total, 17, 430 gene models were represented by probesets that satisfied these criteria. next we asked whether the probeset expression within each gene model was positively correlated for any two cell lines. to do this we calculated the pearson correlation coefficient between the vectors of median signal estimates across replicates in cyt - es versus hcns - scns. the vast majority of genes ( > 80 % ) was found to have probeset - level pearson correlation coefficients of greater than 0. 8 ( figure 3a ). next we randomly permuted the association between the median signal estimates and the probesets for each gene in hescs ( or hcns - scns ) and observed that the distribution of pearson correlation coefficients for the permuted sets was centered at zero, as expected ( figure 3a ). this indicated that the signal estimates for probesets between hescs and hcns - scns were highly correlated and suggested that a scatter plot of probeset signal estimates between hescs and hcns - scns would reveal a linear relationship for the majority of genes.