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).