Description
The EGFR Plus RGQ PCR Kit is a research use only assay for the qualitative detection and identification of mutations in exons 18, 19, 20 and 21 of the epidermal growth factor receptor (EGFR) gene using DNA extracted from plasma or formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The kit is also intended for the semi-quantitative measurement of somatic mutations in exons 18, 20 and 21 of the EGFR gene using DNA samples extracted from plasma. The assay uses real-time PCR on the Rotor-Gene Q 5plex HRM instrument.
The EGFR Plus RGQ PCR Kit provides reagents for real-time PCR amplification reactions to detect 42 EGFR mutations. The assays use mutation-specific reactions involving ARMS (amplification-refractory mutation system) and PCR clamp designs to detect mutations in DNA derived from FFPE tumor tissue and plasma from non-small cell lung cancer (NSCLC). During PCR, if the target of interest is present, both forward and reverse primers specifically anneal and flank the probe. The 5'→3' exonuclease activity of the DNA polymerase cleaves the probe between the reporter and the quencher only if the 3 oligonucleotides hybridize to the target. The probe fragments are then displaced from the target, and polymerization of the strand continues. The 3' end of the probe is blocked to prevent extension of the probe during PCR (see Reaction principle). The increase in fluorescence signal is detected only if the target sequence is complementary to the primers and probe and hence amplified during PCR.
The EGFR Plus RGQ PCR Kit comprises four triplex assays, each containing all the oligonucleotides (primers, probes and oligonucleotide clamp, where applicable) necessary to amplify and detect the targeted EGFR mutations. Manual or automated genomic DNA (gDNA) extraction must first be performed from either FFPE tissue or plasma. When starting from FFPE tissue, manual gDNA extraction is performed with the QIAamp DSP DNA FFPE Tissue Kit (cat. no. 60404). When starting from plasma, cfDNA extraction is performed with the QIAamp DSP Circulating Nucleic Acid Kit (cat. no. 61504). In combination with the QIAsymphony SP instrument, automated gDNA extraction from FFPE samples is performed with the QIAsymphony DSP DNA Mini Kit (cat. no. 937236), while automated cfDNA extraction from plasma samples is performed with the QIAsymphony DSP Circulating DNA Kit (cat. no. 937556). Following gDNA extraction, the DNA is quantified followed by PCR on the Rotor-Gene Q instrument. After the PCR run, analysis and expression calls are performed manually.
