title: Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays.
abstract: Malignant melanoma, the deadliest form of skin cancer, is characterized by a predominant mutation in the BRAF gene. Drugs that target tumours carrying this mutation have recently entered the clinic. Accordingly, patients are routinely screened for mutations in this gene to determine whether they can benefit from this type of treatment. The current gold standard for mutation screening uses real-time polymerase chain reaction and sequencing methods. Here we show that an assay based on microcantilever arrays can detect the mutation nanomechanically without amplification in total RNA samples isolated from melanoma cells. The assay is based on a BRAF-specific oligonucleotide probe. We detected mutant BRAF at a concentration of 500 pM in a 50-fold excess of the wild-type sequence. The method was able to distinguish melanoma cells carrying the mutation from wild-type cells using as little as 20 ng µl(-1) of RNA material, without prior PCR amplification and use of labels.
authors: Swiss Nano Institute, University of Basel, Klingelbergstrasse 82, 4056 Basel, Switzerland. francois.huber@unibas.ch
authors: F
authors: F
authors: Huber
authors: H P
authors: HP
authors: Lang
authors: N
authors: N
authors: Backmann
authors: D
authors: D
authors: Rimoldi
authors: Ch
authors: Ch
authors: Gerber
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods; D003362:Cost-Benefit Analysis; D053938:DNA Fragmentation; D005838:Genotype; D006801:Humans; D008545:Melanoma / Q000175:diagnosis* / Q000235:genetics* / Q000473:pathology; D046228:Microarray Analysis / Q000295:instrumentation / Q000379:methods; D009154:Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D012878:Skin Neoplasms / Q000175:diagnosis* / Q000235:genetics* / Q000473:pathology
chemical_list: D012333:RNA, Messenger; D012313:RNA; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
references: Proc Natl Acad Sci U S A. 2005 Oct 11;102(41):14587-92
references: 16192357
references: Clin Ther. 2012 Jul;34(7):1474-86
references: 22742884
references: N Engl J Med. 2012 Jul 12;367(2):107-14
references: 22663011
references: Biophys Chem. 2000 Jul 31;86(1):37-47
references: 11011698
references: Curr Opin Cell Biol. 2009 Apr;21(2):304-10
references: 19328671
references: Electrophoresis. 2005 Jun;26(13):2553-61
references: 15948220
references: Nat Nanotechnol. 2008 May;3(5):301-7
references: 18654528
references: N Engl J Med. 2011 Jun 16;364(24):2305-15
references: 21663470
references: Anal Chem. 2001 Jan 1;73(1):1-7
references: 11195491
references: Proc Natl Acad Sci U S A. 2002 Jul 23;99(15):9783-8
references: 12119412
references: Science. 2000 Apr 14;288(5464):316-8
references: 10764640
references: Biosens Bioelectron. 2009 Dec 15;25(4):820-5
references: 19765969
references: Nat Nanotechnol. 2006 Dec;1(3):214-20
references: 18654189
references: Clin Exp Metastasis. 2012 Oct;29(7):841-6
references: 22661223
references: Nat Rev Clin Oncol. 2011 May 24;8(7):426-33
references: 21606968
references: Cancer Res. 2003 Sep 15;63(18):5712-5
references: 14522889
references: Anal Chem. 2006 Oct 15;78(20):7104-9
references: 17037909
references: Proc Natl Acad Sci U S A. 1986 Jun;83(11):3746-50
references: 3459152
references: Diagn Mol Pathol. 2012 Mar;21(1):1-8
references: 22306669
references: Biochem Biophys Res Commun. 2010 Aug 27;399(3):313-7
references: 20674547
references: Anal Chem. 2009 Jan 15;81(2):612-7
references: 19086897
references: Curr Top Microbiol Immunol. 2012;355:83-98
references: 21818706
references: Biophys J. 2006 Apr 15;90(8):2970-7
references: 16443650
references: Nature. 2002 Jun 27;417(6892):949-54
references: 12068308
references: Cancer Metastasis Rev. 2005 Jan;24(1):165-83
references: 15785879
references: PLoS One. 2011 Apr 08;6(4):e18369
references: 21494657