A High-Resolution Melting Protocol for Rapid and Accurate Differential Diagnosis of Thyroid Nodules Irene Mancini, Pamela Pinzani, Cinzia Pupilli, Luisa.

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A High-Resolution Melting Protocol for Rapid and Accurate Differential Diagnosis of Thyroid Nodules Irene Mancini, Pamela Pinzani, Cinzia Pupilli, Luisa Petrone, Maria Laura De Feo, Lapo Bencini, Mario Pazzagli, Gianni Forti, Claudio Orlando The Journal of Molecular Diagnostics Volume 14, Issue 5, Pages 501-509 (September 2012) DOI: 10.1016/j.jmoldx.2012.03.003 Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 HRM analysis of cell lines harboring mutations in BRAF, KRAS (codons 12 and 61), HRAS, and NRAS, with HRM profiles and melting curves of control cell lines used as reference during prescreening of FNAB sample. Results are shown for a sample reconstituted from DNA of SK-MEL-28 (BRAF p.V600E, homozygous) mixed with MCF-7 (BRAF wild type) (top left); DNA from CCRF-CEM analyzed for KRAS codon 12 (p.G12D, heterozygous) (top middle) and SW948 for KRAS codon 61 (p.Q61R, heterozygous) (top right); and DNA from CCRF-CEM used as heterozygous control for HRAS gene (p.A59A) (bottom left) and from HT1197 as mutated reference for NRAS codon 61 (p.Q61R, heterozygous) (bottom right). The mutated reference sample for the gene of interest is indicated by an arrow. The Journal of Molecular Diagnostics 2012 14, 501-509DOI: (10.1016/j.jmoldx.2012.03.003) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Examples of hot-spot mutations detected by HRM in FNAB samples. The mutated sample identified by HRM assay is indicated by an arrow. Electropherograms (top right in each panel) confirm the presence of a mutation in the sample with different melting behavior; the nucleotide affected by mutation is bordered by a dotted box. For HRAS and KRAS, mutations caused by different nucleotide substitutions that are clearly detectable by HRM. The Journal of Molecular Diagnostics 2012 14, 501-509DOI: (10.1016/j.jmoldx.2012.03.003) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Examples of rare variants detected by HRM in FNAB samples. The presence of a double mutation in KRAS is easily identified from the abnormal melting profiles of two samples (indicated by arrows), compared with wild-type samples. In electropherograms, arrows indicate the nucleotides affected by adjacent substitutions. The Journal of Molecular Diagnostics 2012 14, 501-509DOI: (10.1016/j.jmoldx.2012.03.003) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions