Real-time PCR quantification of full-length and exon 11 spliced BRCA1 transcripts in human breast cancer cell lines

Germline alterations of the BRCA1 tumor suppressor gene have been implicate d at least in half of familial breast cancers. Nevertheless, in sporadic br east cancer no mutation of this gene has been characterized to date. In spo radic breast tumors, other BRCA1 gene loss of function mechanisms, such as down-regulation of gene expression, have been suggested. In an effort to be tter understand the relationship between BRCA1 expression and malignant tra nsformation, we have adapted the new real-time quantitative PCR method base d on a 5' nuclease assay and the use of doubly labeled fluorescent TaqMan p robes to quantify BRCA1 mRNA. We have compared expression of BRCA1 mRNA wit h or without exon 11 in the normal breast epithelial cell line MCF10a and i n three cancer cell lines (MCF-7, MDA-MB231 and HBL100) by comparing two me thods of quantification: the comparative CT and the standard curve. We foun d that the full length BRCA1 mRNA, which encodes the functional nuclear pro tein, was down-regulated in tumor cells when compared with MCF10a cells. (C ) 2000 Academic Press.