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Quantification of epidermal growth factor receptor T790M mutant transcripts in lung cancer cells by real-time reverse transcriptase-quantitative polymerase chain reaction
Tijdschriftbijdrage - Tijdschriftartikel
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.
Tijdschrift: Anal Biochem
ISSN: 0003-2697
Issue: March
Volume: 398
Pagina's: 266-268
Jaar van publicatie:2010
Trefwoorden:ACQUIRED-RESISTANCE, EGFR MUTATION, GENE MUTATION, GEFITINIB, ASSAY