Quantification of epidermal growth factor receptor T790M mutant transcripts in lung cancer cells by real-time reverse transcriptase-quantitative polymerase chain reaction.
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.
Chen
Gang
G
Laboratory of Molecular Oncology and Department of Medical Oncology, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels, Belgium.
Kronenberger
Peter
P
Umelo
Ijeoma Adaku
IA
Teugels
Erik
E
De Grève
Jacques
J
D000483:Alleles; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000235:genetics; D066246:ErbB Receptors / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology*; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D018076:DNA, Complementary; D012333:RNA, Messenger; D066246:ErbB Receptors