一种用于BRCA1 mRNA的实时定量逆转录聚合酶链反应方法（QRT-PCR）的开发与应用。

Development and applications of a real-time quantitative RT-PCR method (QRT-PCR) for BRCA1 mRNA.

作者信息

Kroupis Christos, Stathopoulou Aliki, Zygalaki Eleni, Ferekidou Lisa, Talieri Maroulio, Lianidou Evi S

机构信息

Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens 15771, Greece.

出版信息

Clin Biochem. 2005 Jan;38(1):50-7. doi: 10.1016/j.clinbiochem.2004.09.012.

DOI:10.1016/j.clinbiochem.2004.09.012

PMID:15607317

Abstract

OBJECTIVES

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.

DESIGN AND 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.

摘要

目的

建立一种用于BRCA1 mRNA的实时定量逆转录聚合酶链反应（RT-PCR）方法，并用其研究人MCF-7乳腺癌细胞在接触抗肿瘤药物和γ射线照射后BRCA1基因的表达情况。

设计与方法

所建立的定量RT-PCR方法基于在LightCycler中进行PCR时对特异于BRCA1 mRNA的荧光素标记TaqMan探针的实时监测。对BRCA1 PCR扩增子进行纯化、定量，并用作已知浓度的标准品，用于该检测方法的建立和分析评估。该方法应用于研究MCF-7人乳腺癌细胞在接触紫杉醇、阿霉素、5-氟尿嘧啶、依托泊苷或γ射线照射后BRCA1基因表达的变化。