Application Guide - QIAxcel Advanced

40 QIAxcel Advanced Application Guide 10/2016 Mutation screening of c-kit and EGFR using the QIAxcel® system prior to Pyrosequencing® Mehran Ghaderi, Department of Pathology and Cytology, Karolinska University Hospital Solna, Stockholm, Sweden The QIAxcel Advanced System was successfully used to separate PCR amplicons and detect mutations in exons of c-kit and EGFR genes found in gastrointestinal stromal tumors (GIST) and non-small cell lung cancers. It reduced analysis time and provided high-quality, reproducible determination of deletions and insertions of key genes. This study shows that the QIAxcel system can be used to increase the efficiency of Pyrosequencing applications. Introduction Screening amplicons for mutations prior to sequencing is a critical step, but it can be tedious and time consuming. It is therefore important to develop rapid and reliable detection techniques that yield results of sufficiently high quality for downstream Sanger sequencing and Pyrosequencing applications. In this study, a method for detecting activating mutations of c-kit and EGFR genes using the QIAxcel Advanced System was assessed. It proved highly suitable for this purpose. Activating mutations of c-kit and EGFR genes can be detected in DNA samples from gastrointestinal stromal tumors (GIST) and non-small cell lung cancers (1–3). Some of the most common mutations are the EGFR exon 19 deletion and exon 20 insertion, and the duplication of the c-kit gene within exon 9 or deletion within exon 11. The assessed QIAxcel-based technique readily detected the mutations in EGFR exon 19 and c-kit exon 11. Materials and methods DNA isolation and purification Genomic DNA was isolated from formalin-fixed paraffin embedded (FFPE) samples with representative tumor cellularity. Sections with a thickness of 10 µm were taken and deparaffinized with xylene. DNA was extracted using the QIAamp® DNA FFPE Tissue Kit. The genomic DNA concentration was measured with a NanoDrop® spectrophotometer (ThermoFisher® Scientific Inc.). * The therascreen EGFR Pyro Kit is not available in the US and Canada.

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