For detecting EGFR mutations and genotyping, a PANAMutyper™ R EGFR kit (Panagene, Daejeon, Korea) and CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) were used. All reactions had a total volume of 25 µL containing 70 ng of template DNA, the primer and PNA probe set along with a PCR master mix. PCR and the melting curve step were performed according to the manufacturer’s protocol. Fluorescence was measured on all four channels (FAM, ROX, Cy5, and HEX) (21 (link),22 (link)).
Panamutyper r egfr kit
Manufactured by Panagene
The PANAMutyper™ R EGFR kit is a laboratory equipment product developed by Panagene. It is designed for the detection and identification of specific genetic mutations in the epidermal growth factor receptor (EGFR) gene. The kit utilizes a real-time PCR-based approach to analyze DNA samples for the presence of these mutations.
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Lab products found in correlation
5 protocols using panamutyper r egfr kit
1
EGFR Mutation Detection and Genotyping
2
Sensitive T790M Mutation Detection
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Circulating cell-free DNA was extracted from 200 μL of plasma samples using the QIAamp MinElute virus spin kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The purity and concentration of extracted DNA were determined by spectrophotometry (NanoDrop 2000, Thermo Fisher Scientific Inc.). DNA samples with absorption ratios of 260/280 nm greater than 1.8 were used for subsequent analyses. For plasma samples, only T790M mutation was analyzed using the PANAMutyper R EGFR kit (Panagene, Daejeon, Korea), a new highly sensitive mutation detection kit that uses a peptide nucleic acid clamping–assisted fluorescence melting curve analysis method in mutation detection and genotyping.
3
Molecular Profiling of Lung Cancer Mutations
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To determine the HER2, EGFR, and KRAS mutation status, DNA was extracted using the DNeasy isolation kit (Qiagen, Valencia, CA, USA) from FFPE tissues according to the manufacturer’s instructions. For the HER2 gene, direct DNA sequencing of exons 18 through 21 was performed in a subset of our cohort (n = 60, comprising cases with driver mutation-negative adenocarcinomas). For EGFR gene analysis, direct DNA sequencing of exons 18 through 21, or utilizing either the PNAClamp™ EGFR Mutation Detection Kit (PANAGENE, Daejeon, Korea) or the PANAMutyper™ R EGFR kit (PANAGENE, Daejeon, Korea), was performed in all cases. For KRAS gene analysis, direct DNA sequencing of codons 12 and 13, or utilizing the PANAMutyper™ R KRAS kit (PANAGENE, Daejeon, Korea) was performed in all cases.
4
Comparative Analysis of EGFR Mutation Detection
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The PANAMutyper R EGFR kit (Panagene, Daejeon, Korea) based on the real-time PCR analysis was used to verify that ULV1 had comparable performance to other platforms in terms of EGFR hotspot mutations. EGFR assays were performed according to the manufacturer’s instructions. For PCR amplification, 5 μL of DNA template, including 34.6 ng of DNA, was added to 19 μL of each master mix and 1 μL of Taq DNA polymerase. Next, PCR reactions were performed using the CFX96 real-time PCR detection system (Bio-Rad Laboratories Inc., Hercules, CA) following the thermal cycling program. Then, amplification and melting curves measured from each fluorescent dye were generated, and the genotype of each sample was determined according to each assay threshold and their melting temperature range. Sample analysis was repeated at least three times, and ULV1 tests were conducted simultaneously using the same samples for direct comparison with PANAMutyper. The agreement between ULV1 and PANAMutyper was assessed using Cohen’s kappa value.
5
EGFR Genotyping in FFPE Tissues
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The tumor samples were prepared as formaline-fixed, paraffin-embedded (FFPE) tissues and tumor DNAs were purified using the TANBead OptiPure FFPE DNA Tube (Taiwan Advanced Nanotech, Taoyuan, Taiwan) according to the manufacturer’s protocol. Then, EGFR genotyping was done through PANAMutyper™ R EGFR kit (Panagene, Daejeon, Korea) according to the manufacturer’s protocol. To prevent the bias, two pathologists read tissue and BALF samples separately in a blinded manner.
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