title: Use of reverse transcription-polymerase chain reaction methodology to detect estrogen-regulated gene expression in small breast cancer specimens.
abstract: We describe the development and use of a sensitive reverse transcription-PCR (RT-PCR) procedure to detect novel estrogen-regulated gene expression in small clinical breast cancer samples, in which such study would be extremely difficult by any other molecular or immunocytochemical means. Assay optimization for pLIV1, estrogen receptors (ERs), progesterone receptors, and pS2 gene products was carried out on 50 primary breast cancers for which comparative Northern analysis and immunocytochemical data were available. Using 27 amplification cycles and a 0.5 microM primer concentration, varying expressions of the gene products were recorded simultaneously with a constant densitometric signal for a coamplified endogenous control gene (alpha-actin). Good concordances were subsequently observed between pLIV1 status generated by RT-PCR and both Northern analysis (P = 0.002) and ER status by immunocytochemistry (P = 0.0244). Agreement was also noted between ER (P = 0.002), progesterone receptor (P = 0.0005), and pS2 (P = 0. 0023) RT-PCR and immunocytochemical methodologies. The RT-PCR assays were then applied to 10 needle core trucut biopsies in which similar relationships were obtained. Our results justify the future use of this RT-PCR methodology to examine new estrogen-regulated genes in small breast cancer samples, and it is envisaged that this technology will prove invaluable in many future breast cancer studies.
authors.affiliation: Tenovus Cancer Research Center and Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XX, United Kingdom.
authors.forename: J M
authors.initials: JM
authors.lastname: Knowlden
authors.forename: J M
authors.initials: JM
authors.lastname: Gee
authors.forename: S
authors.initials: S
authors.lastname: Bryant
authors.forename: R A
authors.initials: RA
authors.lastname: McClelland
authors.forename: D L
authors.initials: DL
authors.lastname: Manning
authors.forename: R
authors.initials: R
authors.lastname: Mansel
authors.forename: I O
authors.initials: IO
authors.lastname: Ellis
authors.forename: R W
authors.initials: RW
authors.lastname: Blamey
authors.forename: J F
authors.initials: JF
authors.lastname: Robertson
authors.forename: R I
authors.initials: RI
authors.lastname: Nicholson
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis* / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology / Q000601:surgery; D004967:Estrogens / Q000502:physiology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D011506:Proteins / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D011960:Receptors, Estrogen / Q000032:analysis* / Q000235:genetics; D011980:Receptors, Progesterone / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014158:Transcription, Genetic; D000071163:Trefoil Factor-1; D025521:Tumor Suppressor Proteins
chemical_list: D014408:Biomarkers, Tumor; D004967:Estrogens; D011506:Proteins; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone; C051838:TFF1 protein, human; D000071163:Trefoil Factor-1; D025521:Tumor Suppressor Proteins