Results

Relative expression of ERC4 messenger RNA in matched normal and 				breast tumor tissues
A recently described triple-primer PCR assay was used to compare the 			 relative expressions of ERC4 messenger RNA between adjacent normal and tumor 			 components [19,24]. In this 			 assay, three primers are used simultaneously during the PCR: the upper primer 			 is able to recognize both WT-ER and ERC4 complementary DNA sequences, whereas 			 the two lower primers are specific for each complementary DNA. Competitive 			 amplification of two PCR products occurs, giving a final PCR product ratio 			 related to the initial input of target complementary DNAs. This approach has 			 been validated previously both by competitive amplification of spiked 			 complementary DNA preparations [19] and by comparison to 			 RNAse protection assays [24].
As shown Figure 1a, two PCR products were 			 obtained, which migrated at the apparent size of 149 and 536 base pairs. These 			 products have been shown to correspond to WT-ER and ERC4 messenger RNAs, 			 respectively [24]. One should note the presence, in 			 samples where WT-ER and ERC4 signals are high (Fig 1a, 			 lane 5), of minor additional bands, one of which has been previously identified 			 as corresponding to exon 2-duplicated ER-α variant complementary DNA 			 [24]. The presence of these minor PCR products did not 			 interfere with the quantitative aspect of the triple-primer PCR assay [24]. For each case, the mean of the ratios obtained in at 			 least three independent PCR experiments, expressed in arbitrary units, is shown 			 for both normal and tumor compartments (Fig 1b). A higher 			 clone 4 messenger RNA relative expression in the tumor compartment was observed 			 in 12 out of 18 cases. This difference did not, however, reach statistical 			 significance (P = 0.47, Wilcoxon signed-rank test). When considering 			 only the ER-positive/PR-positive subset (n = 9), as measured by the 			 ligand-binding assay, a statistically higher ERC4 messenger RNA relative 			 expression was found in the neoplastic components, as compared with matched 			 adjacent normal tissues (P = 0.019, Wilcoxon signed-rank test).

Relative expression of ERD3 messenger RNA in matched normal and 				breast tumor tissues
A PCR assay, performed using primers annealing to sequences in exons 			 2 and 4, was used to investigate ERD3 messenger RNA expression relative to 			 WT-ER in these 18 matched cases. We [18] and others 			 [25] have previously shown that the coamplification of 			 WT-ER and an exon-deleted ER-α variant complemetary DNA resulted in the 			 amplification of two PCR products, the relative signal intensity of which 			 provided a previously validated measurement of exon-deleted ER-α variant 			 expression.
Two PCR products were obtained, that migrated with an apparent size 			 of 354 and 237 base pairs (Fig 2a). These fragments were 			 shown by subcloning and sequencing to correspond to WT-ER and ERD3 messenger 			 RNAs (data not shown). The relative ERD3 signal was measurable in the normal 			 and in the tumor compartments of 13 cases (Fig 2b). Out 			 of these 13 cases, ERD3 messenger RNA expression was higher in the normal 			 compartment in 10 cases. This difference, however, did not reach statistical 			 significance (P = 0.057, Wilcoxon signed-rank test). A significantly 			 higher expression of ERD3 messenger RNA in the normal compared with the 			 adjacent neoplastic components was found when only the ER-positive subset was 			 considered, however (n = 8; P = 0.023, Wilcoxon signed-rank 			 test).

Relative expression of ERD5 messenger RNA in matched normal and 				breast tumor tissues
Using primers annealing to sequences in exons 4 and 6 of WT-ER, we 			 also investigated the relative expression of ERD5 messenger RNA in these 18 			 matched cases. Two PCR products were detected, that migrated at an apparent 			 size of 483 and 344 base pairs, and that have previously been shown to 			 correspond to WT-ER and ERD5 complementary DNAs, respectively (Fig 			 3a). As shown in Fig 3b, a 			 statistically significant higher relative expression of ERD5 messenger RNA was 			 observed in tumor components when this expression was measurable in both normal 			 and adjacent tumor tissues (n = 15; P = 0.035, Wilcoxon 			 signed-rank test).