Mutational Analysis of Thyroid Cancer Genes

We assessed the mutational status of BRAF, RET/PTC1, RET/PTC3, NRAS, HRAS and KRAS in the PTC samples. After a DNase treatment with DNaseI Amplification Grade Kit (Invitrogen, Carlsbad, CA, USA), 1 μg of total RNA was used for reverse transcription using hexamers (3.6 μg μl⁻¹; Roche, Basel, Switzerland) and reverse transcriptase (SuperscriptII RNase H ReverseTranscriptase Kit; Invitrogen). Polymerase chain reactions were performed on 2 μl of cDNA using the recombinant Taq DNA Polymerase Kit (Invitrogen) and appropriate primer pairs (primer sequences and PCR conditions provided in a previous study; Saiselet et al, 2012 (link)). Polymerase chain reaction products were purified with the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer's instructions. Sequencing was performed with the BigDye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) with the sequencer ABI PRISM 3130 (Applied Biosystems) and the genetic analysis program 3130-XI.