Development and applications of a real-time quantitative RT-PCR method (QRT-PCR) for BRCA1 mRNA.
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.
Kroupis
Christos
C
Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens 15771, Greece.
Stathopoulou
Aliki
A
Zygalaki
Eleni
E
Ferekidou
Lisa
L
Talieri
Maroulio
M
Lianidou
Evi S
ES
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
D000970:Antineoplastic Agents; D019313:BRCA1 Protein; D012333:RNA, Messenger; D007041:Hypoxanthine Phosphoribosyltransferase