The PyroMark Q24 system (Qiagen GmbH, Hilden, Germany) was used for pyrosequencing analysis of KRAS and BRAF mutations in DNA from 1 mm formalin-fixed, paraffin-embedded or fresh frozen tumour tissue cores taken from areas with >90 % tumour cells, as previously described [35 (link)]. In brief, DNA was isolated from tumour tissue using QIAamp MinElute spin columns (Qiagen) and DNA regions of interest were PCR-amplified (Veriti 96-Well Fast Thermal Cycler, Applied Biosystems Inc., Foster City, CA, USA).
Detection of mutations in KRAS codons 12 and 13 was performed using Therascreen KRAS Pyro Kit (Qiagen). Analysis of BRAF mutation hotspots in codons 600 and 601 was performed using previously published PCR primers (Richman, JCO 2009) and a novel BRAF sequencing primer (5′-TGATTTTGGTCTAGCTACA-3′) which was designed using the PyroMark Assay Design 2.0 software (Qiagen). All samples with a potential low-level mutation were re-analysed.
Detection of mutations in KRAS codons 12 and 13 was performed using Therascreen KRAS Pyro Kit (Qiagen). Analysis of BRAF mutation hotspots in codons 600 and 601 was performed using previously published PCR primers (Richman, JCO 2009) and a novel BRAF sequencing primer (5′-TGATTTTGGTCTAGCTACA-3′) which was designed using the PyroMark Assay Design 2.0 software (Qiagen). All samples with a potential low-level mutation were re-analysed.
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