B-Raf proto-oncogene serine/threonine kinase (BRAF)V600E gene testing

Determination of the BRAF^V600E gene mutation was carried out for all patients with PTC by PCR techniques (20). DNA was isolated from PTC tissues using a DNA Extraction kit (Promega Corporation, Madison, WI, USA), and BRAF gene exon 15 was detected using a BRAF mutant gene detection kit (Amoy Diagnostics Co., LTD, Fujian, China) according to the manufacturer's protocol and the ABI7500 real-time PCR amplifier (Applied Biosystems; Thermo Fisher Inc., Waltham, MA, USA). The primers for amplification of exon 15 of BRAF were designed as follows: Forward, 5′-TCATAATGCTTGCTCTGATAGGA-3′ and reverse, 5′-GGCCAAAAATTTAATCAGTGGA-3′). All procedures and analyses were carried out in the biomolecular laboratory of the Hongqi Hospital Affiliated to Mudanjiang Medical University.