Dna ffpe tissue kit

Tumor areas (>60%) were dissected under microscopy from 4-μm-thick unstained sections by comparison with an H&E stained slide, and genomic DNA was extracted using a Qiagen DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. After extraction, we measured concentrations and 260/280 and 260/230 nm ratios using a spectrophotometer (ND1000, Nanodrop Technologies, ThermoFisher Scientific, MA, USA). Each sample was then quantified with a Qubit fluorometer (Life Technologies, Carlsbad, CA, USA). Samples of genomic DNA with more than 10 ng measured by the Qubit fluorometer were subjected to AmpliSeq library preparation. To identify actionable CNVs, we used a 21-gene nCounter CNV assay, as previously described [11 (link), 15 (link)]. An AmpliSeq cancer panel v2 was examined.

All mutational analyses were performed after surgical and radioiodine treatments of patients; therefore, the genetic results had no influence on the treatment decisions. DNA samples for molecular analysis were extracted from postoperative surgical specimens using a Qiagen DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. For BRAF T1799A, direct sequencing after conventional polymerase chain reaction (PCR) and mutant enrichment with 3′-modified oligonucleotides-PCR (MEMO-PCR) were performed. DNA sequences from both methods were compared with the normal BRAF gene sequence, specifically exon 15, from the GenBank Database (GenBank accession number NM 004333.4) using sequence assembly software (Gene Codes Corp., Ann Arbor, MI, USA). For TERT C228T, semi-nested PCR was performed to identify TERT promoter mutations. PCR reactions were performed using a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems, Foster City, CA). Cycle sequencing was performed using Big Dye Terminator Cycle Sequencing Ready Reaction kits on an ABI 3730xl Genetic Analyzer (Applied Biosystems). According to the existence of BRAF T1799A or TERT C228T mutations, we categorized patients into three groups as the no mutation group, the BRAF mutation alone group, and the coexistence group of TERT promoter and BRAF mutations (i.e. TERT+BRAF mutation group).