Quantiflour one dsdna system

Genomic DNA (gDNA) was isolated from peripheral blood using Promega (Madison, WI) Maxwell RSC Whole blood DNA kits according to the manufacturer’s instructions. Briefly, 500 µL of the buffy coat or whole blood was added to the RSC extraction cartridge, and 75 µL of nuclease-free water (included in the kit) was added to the elution tube. The cartridge was loaded onto the Maxwell RSC, resulting in isolated gDNA that was ready for downstream applications. Genomic DNA did not undergo any additional purification and was quantitated using the Quantus Fluorometer and Quantiflour ONE dsDNA system by Promega (Madison, WI).

To analyze NRAS and TERT-p mutations, genomic DNA (gDNA) from normal follicular and tumor areas in each case was separately extracted from FFPE tissues by macro-dissection. For cases of NNs with PDc, gDNA of Out-N and PDc areas in the tumor area were separately extracted by microdissection, with a guide slide stained with hematoxylin and eosin. Each FFPE section of 10 µm thickness was dewaxed with 80% xylene in a tube. The dewaxed sample was then washed with absolute ethanol twice and centrifuged at 15,000× g for 15 min at 20 °C. After drying, samples were digested with proteinase K overnight at 56 °C. DNA extraction was performed using the Maxwell RSC DNA FFPE Kit (Promega, Madison, WI, USA) and the Maxwell RSC Instrument (Promega) according to the manufacturer’s protocol. The concentration of double-stranded DNA was quantified by using a QuantiFlour ONE dsDNA system (Promega).