Quantification of epidermal growth factor receptor T790M mutant transcripts in lung cancer cells by real-time reverse transcriptase-quantitative polymerase chain reaction

被引：21

作者：

Chen, Gang ^{[1
,2
,3
]}

Kronenberger, Peter ^{[1
,2
,4
]}

Umelo, Ijeoma Adaku ^{[1
,2
]}

Teugels, Erik ^{[1
,2
]}

De Greve, Jacques ^{[1
,2
]}

机构：

[1] Vrije Univ Brussels, Univ Ziekenhuis Brussel, Mol Oncol Lab, B-1090 Brussels, Belgium

[2] Vrije Univ Brussels, Univ Ziekenhuis Brussel, Dept Med Oncol, B-1090 Brussels, Belgium

[3] Guangxi Med Univ, Dept Pathol, Nanning 530021, Guangxi, Peoples R China

[4] Erasmushogesch Brussel, Dept Gezondheidszorg, Biotechnol Lab, B-1090 Brussels, Belgium

来源：

ANALYTICAL BIOCHEMISTRY | 2010年 / 398卷 / 02期

关键词：

ACQUIRED-RESISTANCE; EGFR MUTATION; GENE MUTATION; GEFITINIB; ASSAY;

D O I：

10.1016/j.ab.2009.11.034

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

A simple and sensitive real-time reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was developed to quantify threonine-to-methionine substitution at amino acid position 790 (T790M) mutant transcripts in a wild-type (wt) epidermal growth factor receptor background. The assay is based on three unmodified oligonucleotides, and both SYBR Green and a Taqman probe can be used. To increase the discrimination between mutant and wt signals, ARMS (amplification refractory mutation system) and LNA (locked nucleic acid) primers were tested, but a benefit was observed only with plasmids and not with cellular complementary DNA. The RT-qPCR assay using transcript-specific primers can detect as few as 1% T790M transcripts in a wt background and, therefore, will be useful in RNA interference studies specifically targeting mutant RNA. (C) 2009 Elsevier Inc. All rights reserved.

引用

页码：266 / 268

页数：3

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