PMC:13912 / 19381-23738 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/13912","sourcedb":"PMC","sourceid":"13912","source_url":"http://www.ncbi.nlm.nih.gov/pmc/13912","text":"Results\n\nRelative expression of ERC4 messenger RNA in matched normal and \t\t\t\tbreast tumor tissues\nA recently described triple-primer PCR assay was used to compare the \t\t\t relative expressions of ERC4 messenger RNA between adjacent normal and tumor \t\t\t components [19,24]. In this \t\t\t assay, three primers are used simultaneously during the PCR: the upper primer \t\t\t is able to recognize both WT-ER and ERC4 complementary DNA sequences, whereas \t\t\t the two lower primers are specific for each complementary DNA. Competitive \t\t\t amplification of two PCR products occurs, giving a final PCR product ratio \t\t\t related to the initial input of target complementary DNAs. This approach has \t\t\t been validated previously both by competitive amplification of spiked \t\t\t complementary DNA preparations [19] and by comparison to \t\t\t RNAse protection assays [24].\nAs shown Figure 1a, two PCR products were \t\t\t obtained, which migrated at the apparent size of 149 and 536 base pairs. These \t\t\t products have been shown to correspond to WT-ER and ERC4 messenger RNAs, \t\t\t respectively [24]. One should note the presence, in \t\t\t samples where WT-ER and ERC4 signals are high (Fig 1a, \t\t\t lane 5), of minor additional bands, one of which has been previously identified \t\t\t as corresponding to exon 2-duplicated ER-α variant complementary DNA \t\t\t [24]. The presence of these minor PCR products did not \t\t\t interfere with the quantitative aspect of the triple-primer PCR assay [24]. For each case, the mean of the ratios obtained in at \t\t\t least three independent PCR experiments, expressed in arbitrary units, is shown \t\t\t for both normal and tumor compartments (Fig 1b). A higher \t\t\t clone 4 messenger RNA relative expression in the tumor compartment was observed \t\t\t in 12 out of 18 cases. This difference did not, however, reach statistical \t\t\t significance (P = 0.47, Wilcoxon signed-rank test). When considering \t\t\t only the ER-positive/PR-positive subset (n = 9), as measured by the \t\t\t ligand-binding assay, a statistically higher ERC4 messenger RNA relative \t\t\t expression was found in the neoplastic components, as compared with matched \t\t\t adjacent normal tissues (P = 0.019, Wilcoxon signed-rank test).\n\nRelative expression of ERD3 messenger RNA in matched normal and \t\t\t\tbreast tumor tissues\nA PCR assay, performed using primers annealing to sequences in exons \t\t\t 2 and 4, was used to investigate ERD3 messenger RNA expression relative to \t\t\t WT-ER in these 18 matched cases. We [18] and others \t\t\t [25] have previously shown that the coamplification of \t\t\t WT-ER and an exon-deleted ER-α variant complemetary DNA resulted in the \t\t\t amplification of two PCR products, the relative signal intensity of which \t\t\t provided a previously validated measurement of exon-deleted ER-α variant \t\t\t expression.\nTwo PCR products were obtained, that migrated with an apparent size \t\t\t of 354 and 237 base pairs (Fig 2a). These fragments were \t\t\t shown by subcloning and sequencing to correspond to WT-ER and ERD3 messenger \t\t\t RNAs (data not shown). The relative ERD3 signal was measurable in the normal \t\t\t and in the tumor compartments of 13 cases (Fig 2b). Out \t\t\t of these 13 cases, ERD3 messenger RNA expression was higher in the normal \t\t\t compartment in 10 cases. This difference, however, did not reach statistical \t\t\t significance (P = 0.057, Wilcoxon signed-rank test). A significantly \t\t\t higher expression of ERD3 messenger RNA in the normal compared with the \t\t\t adjacent neoplastic components was found when only the ER-positive subset was \t\t\t considered, however (n = 8; P = 0.023, Wilcoxon signed-rank \t\t\t test).\n\nRelative expression of ERD5 messenger RNA in matched normal and \t\t\t\tbreast tumor tissues\nUsing primers annealing to sequences in exons 4 and 6 of WT-ER, we \t\t\t also investigated the relative expression of ERD5 messenger RNA in these 18 \t\t\t matched cases. Two PCR products were detected, that migrated at an apparent \t\t\t size of 483 and 344 base pairs, and that have previously been shown to \t\t\t correspond to WT-ER and ERD5 complementary DNAs, respectively (Fig \t\t\t 3a). As shown in Fig 3b, a \t\t\t statistically significant higher relative expression of ERD5 messenger RNA was \t\t\t observed in tumor components when this expression was measurable in both normal \t\t\t and adjacent tumor tissues (n = 15; P = 0.035, Wilcoxon \t\t\t signed-rank test).\n","divisions":[{"label":"Title","span":{"begin":0,"end":7}},{"label":"Section","span":{"begin":9,"end":2196}},{"label":"Title","span":{"begin":9,"end":97}},{"label":"Section","span":{"begin":2198,"end":3612}},{"label":"Title","span":{"begin":2198,"end":2286}},{"label":"Section","span":{"begin":3614,"end":4356}},{"label":"Title","span":{"begin":3614,"end":3702}}],"tracks":[]}