Human breast tissues and reverse transcription
In order to investigate the expressions of ERC4, ERD3 and ERD5 messenger RNA relative to WT-ER messenger RNA within matched normal and breast tumor tissues, eighteen cases were selected in the National Cancer Institute of Canada Manitoba Breast Tumor Bank (Winnipeg, Manitoba, Canada), which had well separated and histopathologically characterized normal and adjacent neoplastic components. The Tumor Bank, which serves as a national Tumor Bank and is funded by the National Cancer Institute of Canada, has been reviewed and received approval from the Ethics Review Committee, Faculty of Medicine, University of Manitoba.
The processing of specimens collected in the Manitoba Breast Tumor Bank has already been described . Briefly, each specimen had been rapidly frozen as soon as possible after surgical removal. A portion of the frozen tissue block was processed to create a paraffin-embedded tissue block that was matched and oriented relative to the remaining frozen block. These paraffin blocks provide high quality histologic sections, which are used for pathologic interpretation and assessment, and are mirror images of the frozen sections used for RNA extractions.
For each case, tumor and adjacent normal tissues from the same individual were histologically characterized by observation of paraffin sections. The presence of normal ducts and lobules, as well as the absence of any atypical lesion, were confirmed in all normal tissue specimens. All tumor components were classified as primary invasive carcinomas. Seven tumors were ER-negative (ER < 3 fmol/mg protein), with progesterone receptor (PR) values ranging from 2.2 to 11.2f mol/mg protein, as measured using ligand-binding assay . Axillary nodal metastases were observed in five of these cases. Eleven tumors were ER-positive (ER values ranged from 3.5 to 159 fmol/mg protein), with PR values ranging from 5.8 to 134 fmol/mg protein. These tumors spanned a wide range of grades (grades 5-9, median 7.5), which were determined using the Nottingham grading system . Axillary nodal metastases were observed in one of these cases. Patients were from 39 to 86 years old (median 54 years). Total RNA was extracted from frozen tissue sections and reverse-transcribed in a final volume of 25 μ l as previously described . The quality of complementary DNAs obtained was assessed by amplification of the ubiquitously expressed glyceraldehyde-3-phosphate dehydrogenase complementary DNA, as described previously .
Triple primer polymerase chain reaction
A previously described triple primer polymerase chain reaction (PCR) assay has been used to coamplify ERC4 and WT-ER-α complementary DNAs [19,24]. Primers used consisted of ERU primer (5' -TGTGCAATGACTATGCTTCA-3', sense, located in WT-ER exon 2, position 792-811), ERL primer (5' -GCTCTTCCTCCTGTTTTTAT-3', antisense, located in WT-ER exon 3, position 940-921), and C4L primer (5' -TTTCAGTCTTCAGATACCCCAG-3', antisense, located in ERC4 sequence, position 1336-1315). The given positions correspond to the published sequences for WT-ER  and ERC4 .
PCR amplifications were performed as previously described [18,24]. Briefly, 0.2 μ l reverse transcription mixture was amplified in a final volume of 15 μ l, in the presence of 1.5 μ Ci of [α-32P] deoxycytidine triphosphate (dCTP; 3000 Ci/mmol), 4 ng/μl of each primer and 0.3 unit of Taq DNA polymerase. Each cycle consisted of 1min at 94°C, 30s at 60°C and 1min at 72°C. PCR products were then separated on 6% polyacrylamide gels containing 7mol/l urea (polyacrylamide gel electrophoresis). After electrophoresis, the gels were dried and autoradiographed. Two PCR products were obtained, which were identified by subcloning and sequencing, performed as previously described . PCR products migrating with the apparent size of 149 and 536 base pairs were shown to correspond to WT-ER and ERC4 complementary DNAs, respectively.
Polymerase chain reaction
Two different primer sets, ERD3 and ERD5, were used to coamplify WT-ER and ERD3 complementary DNAs, and WT-ER and ERD5 complementary DNAs, respectively. ERD3 primer set consisted of D3U primer (5' -TGTGCAATGACTATGCTTCA-3', sense, located in WT-ER exon 2, position 792-811) and D3L primer (5' -TGTTCTTCTTAGAGCGTTTGA-3', antisense, located in WT-ER exon 4, position 1145-1125). ERD5 primer set consisted of D5U primer (5' -CAGGGGTGAAGTGGGGTCTGCTG-3', sense, located in WT-ER exon 4, position 1060-1082) and D5L primer (5'-α TGCGGAACCGAGATGATGTAGC-3', anti-sense, located in WT-ER exon 6, position 1542-1520). The given positions correspond to published sequences for WT-ER .
PCR amplifications were performed and PCR products analyzed as previously described . Briefly, 0.2 μ l reverse transcription mixture was amplified in a final volume of 15 μ l, in the presence of 1.5 μ Ci of [α-32P] dCTP (3000 Ci/mmol), 4ng/μ l of each primer of the primer set considered (ERD3 or ERD5 primer set) and 0.3 unit of Taq DNA polymerase. Each cycle consisted of 30s at 94°C, 30s at 60°C and 30s at 72°C. PCR products were then separated on 6% polyacrylamide gels containing 7mol/l urea (polyacrylamide gel electrophoresis). Following electrophoresis, the gels were dried and autoradiographed. For each PCR, two PCR products were obtained, which were identified by subcloning and sequencing. PCR products migrating with the apparent size of 354 and 483 base pairs, using ERD3 and ERD5 primer set, respectively, were shown to correspond to WT-ER complementary DNA. PCR products migrating with the apparent size of 237 and 344 base pairs, using ERD3 and ERD5 primer set, were shown to correspond to ERD3 and ERD5 complementary DNAs, respectively.
Quantitation and statistical analysis
For each experiment, bands corresponding to the variant messenger RNA (ie ERC4, ERD3 or ERD5) and to WT-ER were excised from the gel and counted in a scintillation counter. For each set of primers (ie ERC4, ERD3 and ERD5 primer set) and for each sample, four independent PCR assays were performed. The ratios between ERC4, ERD3 or ERD5 signals and corresponding WT-ER signal were calculated. For each experiment, in order to correct for overall interassay variations (due to different batches of radiolabelled [α -32P] dCTP or of Taq DNA polymerase), the ratio observed in the same particular tumor (case number 12) was arbitrarily given the value of one and all other ratios expressed relatively. Under our experimental conditions, some samples did not have measurable levels (ie signal lower than twice the background value) of ERD3 or ERD5 variant messenger RNAs (see Figs 2a and 3a) in any of the four repetitions performed. Only cases that had detectable levels in at least three of the replicates in both their normal and tumor compartments were included in the statistical analysis. The significance of the differences in the relative levels of expression of ERC4, ERD3 and ERD5 messenger RNAs between matched normal and tumor components was determined using the Wilcoxon signed-rank test.