Quantification of epidermal growth factor receptor T790M mutant transcripts in lung cancer cells by real-time reverse transcriptase-quantitative polymerase chain reaction.
[en] 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.
Disciplines :
Oncology
Author, co-author :
Chen, Gang
Kronenberger, Peter
Umelo, Ijeoma ; Université de Liège > Département des sciences biomédicales et précliniques > GIGA-R : Labo de recherche sur les métastases
Teugels, Erik
De Greve, Jacques
Language :
English
Title :
Quantification of epidermal growth factor receptor T790M mutant transcripts in lung cancer cells by real-time reverse transcriptase-quantitative polymerase chain reaction.
Publication date :
2010
Journal title :
Analytical Biochemistry
ISSN :
0003-2697
eISSN :
1096-0309
Publisher :
Elsevier, Atlanta, United States - Florida
Volume :
398
Issue :
2
Pages :
266-8
Peer reviewed :
Peer Reviewed verified by ORBi
Commentary :
Copyright (c) 2009 Elsevier Inc. All rights reserved.
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