Differential expression of EDN3 mRNA in human breast cancer. (a) A multiple tissue Northern blot hybridised with an EDN3-specific probe indicates ubiquitous expression of EDN3 mRNA in a variety of normal tissue types. In some tissues, both major EDN3 transcript variants are expressed (2.4 and 2.7 kb). (b) The same probe was hybridised to a breast cancer array containing cDNA pairs from 50 breast carcinoma tissues (T), 50 matched normal breast tissues (N) and 3 metastatic tissues. The outlined groups represent matched pairs, including the metastatic deposit, and consecutive numbers indicate specimen spots on the array. (c) Box plot demonstrating significant downregulation of EDN3 expression between normal and tumourous breast tissues (P < 0.001, Student paired t test; extreme value of specimen #29 omitted). Horizontal lines indicate group medians, and boxes indicate 25% to 75% quartiles, range, peak and minimum. (d) Real-time polymerase chain reaction analysis demonstrated loss of EDN3 expression in 78% of breast carcinomas by FC2 (fold change of greater than 2) (black bars) whilst 22% showed no deregulation (grey bars). (e) Box plot demonstrating the different distributions of EDN3 expression among normal breast tissues and breast carcinomas (P < 0.001, Mann-Whitney U test). Horizontal lines indicate group medians, and boxes indicate 25% to 75% quartiles, range, peak and minimum. (f) Fold changes of EDN3 expression in 17 matched pairs of normal breast and breast carcinoma samples revealed loss of expression in 13 cases (76%) and a median expression change of 13-fold. EDN3, endothelin-3.