Nras pyro kit

DNA was extracted from unstained paraffin-embedded tissue sections of surgical resection specimens using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) after manual microdissection. After PCR using primers targeting codon 61 using the PyroMark PCR Kit (Qiagen), pyrosequencing was performed according to standard procedures using the NRAS Pyro Kit (Qiagen) and PyroMark Q24 System (Qiagen). Program outputs were analyzed with the PyroMark Q24 software (version 2.0.6; Qiagen) using allele quantification mode. The NRAS mutation status was considered positive if the mutant allele percentage was 5% or more.

KRAS and NRAS mutations were analyzed in 1 area of both lesions in SP-CRC patients, as well as in the LNs and systemic metastatic lesions. First, KRAS codons 12 and 13 were tested, and if they were wild-type, mutations in KRAS codon 61 and NRAS codons 12, 13, and 61 were examined. If the sample remained wild-type for the tested codons, then, KRAS and NRAS codons 117 and 146 were analyzed.
Mutations were evaluated by pyrosequencing per the manufacturer's instructions [KRAS PyroMarkTM Q24 kit, NRAS Pyro Kit, RAS extension KIT (Qiagen)]. Ten microliters of biotinylated PCR product was conjugated to streptavidin-sepharose beads (GE Healthcare) per a standard protocol for single-strand preparation. Pyrosequencing was performed using the PyroMarkTM Gold Q24 reagent kit (Qiagen). A cutoff value of 5% was used to define a case as positive.

After surgery, four pathologists examined the thyroid sections stained with hematoxylin and eosin. In one case HBME-1 staining, which is considered a good marker of malignancy [16 (link), 17 (link)], was also performed and evaluated.
Although molecular biology analyses had already been performed on the FNA of all Thy3 nodules before RFA, they were repeated on the surgically resected nodules. BRAF mutational status (including V600E/Ec, V600D, 600K, and V600R mutations) was evaluated by Therascreen BRAF RSQ PCR kit (Qiagen, UK). NRAS mutations were analysed by NRAS Pyrokit for mutations in codons 12, 13, 56, 61, 117, and 146 (Qiagen, UK). DNA was extracted from paraffin blocks and tested by using real-time polymerase chain reaction on the Rotor-Gene Q MDx 5plex HRM instrument (Qiagen, UK) and on the PyroMark Q24 System (Qiagen, UK), respectively.