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 [21]. 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 			 [22]. 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 [23]. 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 [18]. The quality 			 of complementary DNAs obtained was assessed by amplification of the 			 ubiquitously expressed glyceraldehyde-3-phosphate dehydrogenase complementary 			 DNA, as described previously [18].