Amoydx ffpe dna kit

Genomic DNA isolated from the primary PTC was extracted from paraffin-embedded tissues. Sections with a confirmed tumor were deparaffinized and collected for DNA extraction. The process was performed using a spinal column procedure (AmoyDx^® FFPE DNA Kit, Amoy Diagnostics, China) according to the manufacturer’s instructions. The absorbances of the DNA samples were measured with a spectrophotometer, and the A₂₆₀/A₂₈₀ values were all between 1.8 and 2.0. The DNA samples were stored at –20°C until real-time qualitative PCR analysis. The BRAF^V600E mutation status of each primary PTC was determined using the AmoyDx^® BRAF^V600E Mutation Detection Kit (Amoy Diagnostics, China). The mutant BRAF gene (encoding BRAF^V600E) was amplified with specific primers and was detected with novel probes using Bio-Rad CFX96 (Bio-Rad Laboratories, USA) according to the manufacturer’s instructions. The carboxyfluorescein (FAM) fluorescence signal was used to evaluate the mutation status of the sample. When the sample FAM Ct value was ≥ 28, the sample was classified as negative or below the detection limit of the kit. When the sample FAM Ct value was < 28, the sample was classified as mutation positive.

All surgical tissue specimens were fixed with formaldehyde and embedded with paraffin. Three to five sections were taken to scrape the tissue in the rich region of tumor cells, as compared with the HE sections. DNA extraction was conducted in accordance with the instructions (Amoydx ^® FFPE DNA Kit, Amoy Diagnostics, Xiamen, China). TP53 status was evaluated by PCR amplification and DNA sequencing. The reaction system of PCR amplification, including target DNA 1 μL, 2×PCR reaction buffer 12.5 μL, each primer (10 μM) 2 μL, and ddH₂O, was supplemented at 25 μL. Cyclic parameters were as follows: pre-denaturation at 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, for a total of 31 cycles; and extension at 72°C for 5 min. The amplified product was electrophoresis in a 1.5% agarose gel. The amplified products were recovered by gel-cutting and identified by agarose gel electrophoresis. The bidirectional sequencing analysis was conducted by Sanger sequencing method with ABI3500DX gene sequencer. This method was currently the accepted standard for identifying TP53 mutations.

The results of BRAF V600E mutation test using preoperative qPCR were recorded.
In preoperative qPCR, DNA extraction was performed on fine-needle cytologic samples using the AmoyDx^® FFPE DNA Kit (Amoy Diagnostics Co., Ltd., Xiamen, China) according to the manufacturer’s protocols. The concentration and purity of extracted DNA were assessed by Nanodrop spectrophotometry.
BRAF V600E mutation detection was performed in the Cellular and Molecular Diagnostic Centre of Sun Yan-Sen Memorial Hospital, Sun Yat-Sen University. DNA from the 423 patients was tested using the AmoyDx^®BRAF Mutations Detection Kit (Amoy Diagnostics Co., Ltd., Xiamen, China) under the principle of the amplification refractory mutation system (ARMS), detecting BRAF V600E mutation (exon 15). AmoyDx^®BRAF Mutations Detection Kit was used to detect BRAF V600E mutation in cytological specimens and tissue specimens in some previous studies (16 (link), 17 (link)). Briefly, the PCR was carried out on a 7500 Real‐Time PCR System (Applied Biosystems) according to the manufacturer’s protocol with 10 ng of DNA in each reaction system. The PCR kit allows an LOD (limit of detection) as low as 1% for BRAF V600E (PCR kit instructions). All results were confirmed according to the criterion suggested by the manufacturer.