Development and applications of a real-time quantitative RT-PCR method (QRT-PCR) for BRCA1 mRNA
Development and applications of a real-time quantitative RT-PCR method (QRT-PCR) for BRCA1 mRNA
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DOI：
10.1016/j.clinbiochem.2004.09.012
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发表时间：
2005-01-01
影响因子：
2.8
通讯作者：
Lianidou, ES
中科院分区：
文献类型：
--
作者：
Kroupis, C;Stathopoulou, A;Lianidou, ES
目的：建立BRCA 1 mRNA的实时定量RT-PCR方法，并将其应用于人乳腺癌MCF-7细胞暴露于抗肿瘤药物和γ射线后BRCA 1基因表达的研究。设计和方法：所建立的QRT-PCR方法是基于实时荧光素标记的TaqMan探针的实时监测，该探针特异于BRCA 1 mRNA，在PCR过程中在LightCycler中。对BRCA 1 PCR扩增子进行纯化、定量，并用作试验开发和分析评价的已知浓度标准品。该方法被应用于研究暴露于紫杉醇，阿霉素，5-氟尿嘧啶，足叶乙甙或γ射线照射后BRCA 1基因表达的改变在人MCF-7乳腺癌cells.Results：所开发的方法是定量的，高度特异性的mRNA和高度敏感性（检测限为4 BRCA 1拷贝每马克杯的总RNA）。我们观察到的BRCA 1表达的减少，而γ射线照射的MCF-7细胞有一个高峰，在10戈伊dose.Conclusions：BRCA 1的定量RT-PCR方法是定量的，高度敏感和特异性。所提出的方法是快速，自动化和成本效益，并可用于研究BRCA 1在各种临床样本中的表达。(C)2004年加拿大临床化学家协会。All rights reserved.
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 antieoplastic 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. (C) 2004 The Canadian Society of Clinical Chemists. All rights reserved.