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AmoyDx BRAF V600E Mutation Detection Kit

Manufactured by Amoy Diagnostics
Sourced in China

The AmoyDx BRAF V600E Mutation Detection Kit is a real-time PCR-based diagnostic assay designed to detect the BRAF V600E mutation in human genomic DNA samples. The kit provides a qualitative analysis of the presence or absence of the BRAF V600E mutation.

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5 protocols using AmoyDx BRAF V600E Mutation Detection 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 BRAFV600E mutation status of each primary PTC was determined using the AmoyDx® BRAFV600E Mutation Detection Kit (Amoy Diagnostics, China). The mutant BRAF gene (encoding BRAFV600E) 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.
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KRAS, NRAS and BRAF mutation status of the cell lines was determined using the Broad Institute’s Cancer Cell Line Encyclopedia (www.broadinstitute.org/ccle/home). KRAS (NCBI Gene ID: 3845) and BRAF (NCBI Gene ID: 673) hotspot mutation status was confirmed using DxS TheraScreen KRAS PCR Kit (Qiagen) and AmoyDx BRAF V600E Mutation Detection Kit (Amoy Diagnostics), respectively.
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The BRAFV600E mutation analysis was performed with DNA that extracted from remaining FNA cells after cytologic evaluation. Real-time PCR was performed using the CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA). The mutant BRAF gene (encoding BRAF V600E) was amplified with specific primers. Thermal cycling conditions were initial denaturation of 1 cycle for 5 minutes at 95 °C, 95 °C for 10 minutes (1 cycle), and 95 °C for 15 seconds, followed by 15 cycles of 95 °C for 25 s, 64 °C for 20 s, and 72 °C for 20 s with a final step of annealing and elongation of 31cycles at 93 °C for 25 s, 60 °C for 35 s and 72 °C for 20 s. The BRAF V600E mutation status of each primary PTC was determined using the AmoyDx BRAF V600E Mutation Detection Kit (Amoy Diagnostics). The FAM signals of the mutation detection system indicate the mutation status of the sample. The HEX/VIC signals indicate the internal control status. The FAM Ct value was checked for each sample: a) If the sample FAM Ct value ≥28, the sample was classified as negative or below the detection limit of the kit. b) If the sample FAM Ct value < 28, the sample was classified as mutation positive.
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Genomic DNA isolated from paraffin-embedded tissues of PTC patients. The paraffin-embedded tissue blocks of tumor tissue was sectioned for hematoxylin and eosin staining with the purpose of identifying pathological diagnosis. Regions with neoplastic compositions of 80% or greater were marked as tumor tissue under optical microscope. Depending on the size of the tissue, 3–4 pieces of 5-μm-thick sections of each block were manually microdissected using a needle and collected into 1.5 ml microtubes for DNA extraction. The Genomic DNA isolation process was performed using the Puregene Tissue Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. The absorbance of the DNA samples was measured using a spectrophotometer, and only the A260/A280 values between 1.8 and 2.0 were accepted for the next step. The extracted DNA was stored at – 80 °C for next procedure. The status of the BRAFV600E mutation of each sample was examined by qRT-PCR using the AmoyDx BRAFV600E Mutation Detection Kit (Amoy Diagnostics, Xiamen, China), and the presence of the mutation was evaluated following the manufacturer’s instructions. The sample was classified as mutation-positive when the cycle threshold (Ct) was ≤ 28 and as negative when Ct was > 28.
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5

BRAF V600E Mutation Detection in Papillary Thyroid Carcinoma

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 manufacturer instructions. The absorbance of the DNA samples was measured with a spectrophotometer, and the A 260 /A 280 values were all between 1.8 and 2.0. The DNA samples were stored at -20°C until real-time qualitative polymerase chain reaction analysis. The BRAF V600E mutation status of each primary PTC was determined using the AmoyDx BRAF V600E Mutation Detection Kit (Amoy Diagnostics). The mutant BRAF gene (encoding BRAF V600E) was amplified with specific primers and detected with novel probes using Bio-Rad CFX96 (Bio-Rad Laboratories, USA) according to manufacturer instructions. The 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 being below the detection limit of the kit. When the sample FAM Ct value was <28, the sample was classified as mutation-positive.
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