Because the results between the 2 centrifugation groups were inconsistent in some cases, the results related to EGFR mutation in cfDNA were compared with those obtained following a recently performed tissue biopsy or from pleural fluid. Paraffin-embedded samples from tissue biopsy were placed onto slides to prepare 4-μm-thick sections. One slide of the block was stained with H&E and reexamined for the presence of cancer cells. The enriched area was marked by a pathologist to validate tumor purity of 25% or more. These cancer cell–enriched areas were microdissected, and DNA was extracted. EGFR mutations were analyzed using a PNAClamp EGFR Mutation Detection Kit or PANAMutyper (PANAGENE) on a Bio-Rad CFX96 system in the pathology laboratory.
Panamutyper
Manufactured by Panagene
The PANAMutyper is a laboratory equipment product designed for the analysis and detection of genetic mutations. It utilizes a proprietary mutation detection technology to identify specific genetic variants within a sample. The core function of the PANAMutyper is to provide accurate and reliable results for genetic analysis applications.
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5 protocols using panamutyper
1
Validating EGFR Mutation in cfDNA
2
EGFR Mutation Detection Protocol
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EGFR mutation tests were performed using tumor tissues. Genomic DNA was extracted from formalin-fixed, paraffin-embedded, 5-µm-thick tissue sections using the High Pure Template Preparation Kit (Roche Applied Science, Mannheim, Germany). The extracted DNA was stored at –20 ˚C until analysis. PANAMutyper™ (PANAGENE Inc., Daejeon, South Korea), a PNA-Clamping-based EGFR mutation detection kit, was used for the detection of EGFR mutations. The primer sets covered mutations or deletions spanning exons 18–21 of the genes encoding the tyrosine kinase domain of EGFR. Results were interpreted according to the manufacturer’s instructions.
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EGFR mutation tests were performed using tumor tissues. Genomic DNA was extracted from formalin-fixed, paraffin-embedded, 5-µm-thick tissue sections using the High Pure Template Preparation Kit (Roche Applied Science, Mannheim, Germany). The extracted DNA was stored at –20 ˚C until analysis. PANAMutyper™ (PANAGENE Inc., Daejeon, South Korea), a PNA-Clamping-based EGFR mutation detection kit, was used for the detection of EGFR mutations. The primer sets covered mutations or deletions spanning exons 18–21 of the genes encoding the tyrosine kinase domain of EGFR. Results were interpreted according to the manufacturer’s instructions.
3
EGFR Mutation Detection in Tumor Samples
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EGFR mutation testing was performed on the tumor samples. Sections with 5-μm-thickness were prepared from formalin-fixed, paraffin-embedded tissue, and genomic DNA was extracted using the High Pure Template Preparation kit (Roche Applied Science, Mannheim, Germany) and was stored at -20°C until analysis. PANA Mutyper™ (PANAGENE Inc., Daejeon, South Korea), a PNA-clamping-based EGFR mutation detection kit, was used to detect the mutations. The primer sets included mutations or deletions in exons 18-21 that encode the tyrosine kinase domain of EGFR. The results were interpreted according to the manufacturer's instructions.
4
Real-Time PCR-Based Mutation Detection
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PANAMutyper ® (PANAGENE, Korea) contains a PNA clamp and PNA detection probes in each reaction tube. All reactions were done in 25-μL volumes using 5-μL template DNA, 19-μL primer and PNA probe master mix set, and 1-μL taq polymerase per sample. All reagents used were included in the kit. Real-time PCR reactions were performed using a CFX 96 ® (Bio-Rad, CA, USA). PCR was performed under the following conditions: 50°C for 2 min and 95°C for 15 min as two holding periods; 15 cycles of 95°C for 30 s, 70°C for 20 s, 63°C for 60 s; 35 cycles of 95°C for 10 s, 53°C for 20 s, 73°C for 20 s; and a melting curve step (from 35°C to 75°C with gradual increment for 0.5°C for 3 s). Fluorescence was measured on all the four channels (FAM, ROX, Cy5, and HEX). Melting peaks were derived from the melting curve data. Mutations were detected by the melting temperature of each tube for each fluorescent dye.
5
Detecting EGFR and KRAS Mutations
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To detect epidermal growth factor receptor (EGFR) mutations in exons 18-21 and KRAS mutations in codons 12, 13, and 61, EGFR and KRAS were analyzed at the time of diagnosis using a PCR-based assay (PANAMutyper, Panagene, Daejeon, Korea) according to the protocol provided by the manufacturer.
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