title: Development and applications of a real-time quantitative RT-PCR method (QRT-PCR) for BRCA1 mRNA.
abstract: OBJECTIVE
  To develop a real-time quantitative RT-PCR method for BRCA1 mRNA and then use it for the study of BRCA1 gene expression in human MCF-7 breast cancer cells after their exposure to antineoplastic agents and gamma irradiation.
  
  
  METHODS
  The developed QRT-PCR method is based on the real-time monitoring of a fluorescein-labeled TaqMan probe, specific for BRCA1 mRNA, during PCR in the LightCycler. A BRCA1 PCR amplicon was purified, quantitated and used as a standard of known concentration for the development and analytical evaluation of the assay. The method was applied to study the alteration of BRCA1 gene expression after exposure to taxol, doxorubicin, 5-fluorouracil, etoposide or gamma irradiation in human MCF-7 breast cancer cells.
  
  
  RESULTS
  The developed method is quantitative, highly specific for mRNA and highly sensitive (detection limit of 4 BRCA1 copies per mug of total RNA). We observed a reduction of BRCA1 expression for all antineoplastic agents used, while the gamma irradiated MCF-7 cells had an increase of expression with a peak at the 10 Gy dose.
  
  
  CONCLUSIONS
  The developed BRCA1 QRT-PCR method is quantitative, highly sensitive and specific. The proposed method is rapid, automated, and cost effective and can be used to study BRCA1 expression in a variety of clinical samples.
authors.affiliation: Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens 15771, Greece.
authors.forename: Christos
authors.initials: C
authors.lastname: Kroupis
authors.forename: Aliki
authors.initials: A
authors.lastname: Stathopoulou
authors.forename: Eleni
authors.initials: E
authors.lastname: Zygalaki
authors.forename: Lisa
authors.initials: L
authors.lastname: Ferekidou
authors.forename: Maroulio
authors.initials: M
authors.lastname: Talieri
authors.forename: Evi S
authors.initials: ES
authors.lastname: Lianidou
mesh_terms: D000595:Amino Acid Sequence; D000970:Antineoplastic Agents; D019313:BRCA1 Protein / Q000235:genetics*; D001483:Base Sequence; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D002277:Carcinoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D005260:Female; D005720:Gamma Rays; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D019313:BRCA1 Protein; D012333:RNA, Messenger; D007041:Hypoxanthine Phosphoribosyltransferase