quantitative rt - pcr ( qpcr ) was used to verify the fold change of the expression of 4 genes, including thbs1, mmp3, tnfrsf11b and krtha4 ( figure 3c ). further confirmation can also be done using immunocytochemistry, as antibod ies against these genes are commercially available. figure 3human fibroblast feeder cells can be disting uished from hescs and ebs. bead array identified lists of genes that were uniquely expressed in human fibroblast feeders as compared to hescs ( a ) and hebs ( b ). the four genes whose expression was confirmed by qpcr ( c ) were in bold. in the graph ( c ), gene expression of each gene in feeder cells was designated as 1 fold and the bars represented fold decrease for each gene. thus this comparison allowed us to disting uish between hescs and human feeders and identify candidate markers that could detect feeder cell contamination should human feeders be used in the propagation of hescs. hescs and ebs can be disting uished from each otherillumina bead array analysis confirmed that hescs could be readily disting uished from ebs by global analysis. this raised the possibility that specific subsets of markers could be identified. we and others have used mpss and est scan and generated array data to make lists of hesc - specific genes [ 5, 9, 10, 20 ]. as discussed above, most hesc markers identified by mpss have been detected in the present bead array analysis ( table 3 ), confirming the utility of these previously identified markers for use in assessing undifferentiated status of hescs. in addition, we have generated a list of genes differentially expressed at higher level in ebs than in hescs, a subset of which is shown in table 5. these markers were common to all eb samples tested and included genes known to be expressed in ectoderm, endod erm and mesoderm. the entire set of differentially expressed genes is provided in additional file 3. thus, the bead array format, which allows multiple pairwise comparisons, can be used to identify genes that are expressed by all differentiating eb samples in the present study. our data suggested that a core set of limited markers might be sufficient to monitor the process of differentiation. by suitable selection of different germ cell layer specific markers one may also assess the overall quality of differentiation toward germ cells. table 5genes which are differentially expressed at higher levels in ebs than in hescssymbolall eball eseb / esreln11120. 52224. 0sst1394. 25. 5253. 5slc40a11210. 19. 1133. 0igf24896. 938. 4127. 5sln2017. 817118. 7dcn7588. 176. 299. 6anxa84048. 941. 996. 6aqp11665. 420. 581. 2apob1256. 415. 879. 5ahsg1414. 320. 868. 0nid21476. 731. 447. 0fgb221052. 442. 2lum8439. 4205. 441. 1mgp2772. 469. 839. 7thbd1206. 434. 435. 1serpina11255. 643. 728. 7hand112294437. 428. 1hbe11106. 542. 626. 0ttr7661. 2347. 122. 1hbg21601. 185. 618. 7col2a11636. 291. 817. 8kiaa09771370. 87817. 6afp8941552. 316. 2col3a11355796714. 0igfbp38446. 3603. 414. 0pax61577. 8118. 213. 3apoa19398. 1709. 813. 2frzb3523. 7315. 711. 2spon21548. 7159. 49. 7cebpd1100. 2122. 79. 0dlk13355. 8374. 89. 0rdc11589. 7192. 18. 3bmp41851. 5227. 38. 1pitx21057. 9131. 28. 1acta25045629. 88. 0gas11215. 61547. 9agtrl11053. 8135. 17. 8col5a15876. 4765. 47. 7cdkn1c3134. 1412. 97. 6cxcl142220. 5312. 67. 1dok41011. 1145. 27. 0arhgdib1635. 3246. 56. 6flrt21879. 5314. 36. 0msx12771. 8499. 75. 5 smaller but distinc t differences among undifferentiated hesc linesour cluster analysis indicated that bg01, bg02 and bg03 cell lines were overall more similar to each other than to other lines ( figure 1 and 2 ), but nevertheless showed additional differences than technical or biological repeats of the same sample. this raised the possibility that this microarray strategy may be sufficiently sensitive to identify relatively cell type specific candidate genes that could be used to disting uish one hesc population from another or to identify differences that were due to varied isolation and growth conditions. as a test we looked for differences between bg01, bg02 and bg03, which were grown in the same laboratory under the same conditions. lists of candidate genes are shown in figure 4a, c and 4e and the comparison of these three lines are shown in scatter plots in figure 4b, d and 4f. figure 4bg lines show small but distinc t differences as assessed by bead array. these three hesc lines share high similarities as shown by the scatterplots of bg01 vs bg02 ( b ), bg01 vs bg03 ( d ) and bg02 vs bg03 ( f ). comparisons of all three lines were made and lists of selected genes that were specifically expressed in bg01 ( a ), bg02 ( c ) and bg03 ( f ) are shown. correlation coefficients ( r2 ) were generated using all genes with expression level > 0 ( black and blue dots ), or all genes with detection confidence > 0. 99 ( blue dots ). genes outside the two thin red lines were detected at > 2. 5 - fold difference. we reasoned as well that such a global comparison should allow us to disting uish between male and female lines if genes present on the y chromosome were expressed at high levels in the undifferentiated state and were detected by the bead array. several such candidate genes were identified. the most robust were rps4y, rps4y2, and eif1ay ( figure 5 ). to confirm that these were useful markers, we designed rt - pcr primers and tested their expression in a male ( bg01 ) and a female ( bg03 ) line ( figure 5b ). we noted that several of these continued to be expressed at high levels as escs differentiated to form ebs and upon further differentiation ( data not shown ), suggesting that these markers might be used in adult stem cell and germ cell populations as well. figure 5male and female hesc lines can be disting uished by genes identified by bead array. five potential genes rps4y, rps4y2, eif1ay, vcy, and amely are located in the y chromosome. by comparing the expression level of these genes in all hesc lines, we have found that 3 out of 5 were specifically expressed in male hesc lines i6, bg01 and bg02 ( a ) and this was verified by rt - pcr in male line bg01 and female line bg03 ( b ). g3pdh was used as an internal control. *: represents the gene expression level is detected at < 0. 99 confidence. in summary, our data suggest that the bead array format is sufficiently sensitive and global that it can disting uish one cell line from another even if those two cell lines are grown in the same laboratory under virtually identical conditions. bead array can also be used to disting uish between male and female lines. comparison of diploid pluripotent cells with ntera2 and bg01 variantour previous results have suggested that ec lines share many of the properties of hescs and can be used as a useful model for initial testing of biological questions [ 21 ]. more recently we have identified bg01v as a karyotypically abnormal variant that behaves much like its normal counterpart bg01, but is not subject to the same constraints of use as karyotypically normal hescs [ 13 ]. given the sensitivity of the bead array analysis, we tested its ability to detect the overall similarities and differences between ntera2 and a pooled esc sample or between the karyotypically abnormal bg01v and its normal parent line ( figure 6 ). figure 6diploid pluripotent ec cell line ntera2 and karyotypically abnormal hesc line bg01v can be disting uished from normal hescs using illumina array. comparison of ntera2 and pooled hesc sample resulted a correlation coefficient of 0. 8997. two lists of genes, which were specifically expressed in ntera2 ( c ) or in hescs ( e ) were identified. likewise, while sharing similarities with bg01 ( b, correlation coefficient = 0. 9043 ), bg01v was different from bg01 in expression for many genes, particularly genes from the tgfβ pathway ( d, f ). black dots represent genes that were detected at > 0 expression level, blue dots represent genes that were detected both at > 0 expression level and at > 0. 99 confidence.