years) and 4 grossly normal colon (2 female, 2 male, av- erage age 65 years) FFPE blocks were purchased (Advanced Tissue Services). mCRC samples were classi- fied as KRAS mutation posi- tive by the vendor. Samples were prepared using stan- dard protocols (QIAGEN)
• Droplet Digital PCR (ddP- CR™) was performed on 1–5 µl per sample per well using either a multiplexed KRAS G12/G13 Assay or val- idated PrimePCR™ ddPCR Mutation Assay for one of seven individual KRAS mu- tations (G12D, G12V, G13D, G12A, G12C, G12R, G12S, Bio-Rad)
• Positive mutation refer- ences were from Horizon Diagnostics, and negative controls were wild-type–only from Promega Corporation (female genomic DNA [gDNA]). Statistical signifi- cance was determined using 95% confidence intervals
Conclusions
• We have demonstrated sensitive and precise detec- tion (less than 1%, single re- action) of multiple actionable KRAS mutations in FFPE samples from patients with colorectal cancer
• Concordance between du- plex- and multiplex-based detection is excellent
• Droplet Digital PCR provides a simple and robust workflow for mutation detection of patient samples in a rapid and cost-effective manner • UDG treatment of FFPE DNA reduces the false positives generated by deaminated C>T transitions caused by formalin fixation
References
Do H and Dobrovic A (2012). Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil- DNA glycosylase. Oncotarget 3, 546–558.
Faulkner NE et al. KRAS mutation analyses of more than 16,500 colorectal carcinomas. Poster presented at: 2010 ASCO-NCI-EORTC Annual Meeting on Molecular Markers in Cancer; October 18–20, 2010; Hollywood, Florida [unpublished data].
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bio-rad.com/web/ddPCRKRAS for more information. FAM is a trademark of Applera Corporation. BioTechniques 58:270-271 (May 2015) doi 10.2144/000114293
Vol. 58 | No. 5 | 2015 271
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