Therascreen egfr pyro kit

Manufactured by Qiagen

Sourced in Germany

The Therascreen EGFR Pyro Kit is a laboratory equipment product designed for the detection and analysis of mutations in the EGFR gene. It utilizes pyrosequencing technology to provide a qualitative assessment of specific EGFR mutations.

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Lab products found in correlation

Qiaamp dna ffpe tissue kit, by Qiagen (2 mentions) Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions) Pyromarktm q24, by Qiagen (1 mentions) High pure pcr template preparation kit, by Roche (1 mentions) Pyromark q24 system, by Qiagen (1 mentions) Bioclear, by Bio-Optica (1 mentions) Agilent 2200 tapestation system, by Agilent Technologies (1 mentions) Genomic dna screentape assay, by Agilent Technologies (1 mentions) Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions) Taq polymerase, by Takara Bio (1 mentions) Oncomine focus assay, by Thermo Fisher Scientific (1 mentions) Cobas egfr mutation test version 2, by Roche (1 mentions)

4 protocols using therascreen egfr pyro kit

EGFR Mutation Analysis in LADC

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Tissue samples were acquired during diagnostic procedures including wedge resection surgery and bronchoscopic- or transthoracic needle biopsy. The diagnosis of LADC of each case was confirmed on a freshly prepared hematoxylin and eosin stained slide. As this estimate is critical for the study, the exact proportion of neoplastic cells was reassessed by two independent expert histopathologists. All mutational analyses were performed at the 1^st Department of Pathology and Experimental Cancer Research of the Semmelweis University. Genomic DNA was extracted using the High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland) in accordance with the manufacturer’s instructions, and all samples underwent testing for mutation in EGFR codon (in exon 18, 19, 21) using the Therascreen EGFR Pyro Kit (Qiagen, Germany) on a PyroMark^TM Q24 (Qiagen) pyrosequencing instrument. The percentage of mutated nucleic acid was calculated with the equipment software (Qiagen PyroMark^TM), relating the peak of mutated base to that of the wild-type base, which was considered 100%. The obtained VAF for each patient was then normalized to the proportion of neoplastic cells in each specimen using the following formula:
Where VAF represents the percentage of the EGFR variant alleles determined by the pyrosequencing assay and TC% is the estimated percentage of neoplastic cells.

Gieszer B., Megyesfalvi Z., Dulai V., Papay J., Kovalszky I., Timar J., Fillinger J., Harko T., Pipek O., Teglasi V., Regos E., Papp G., Szallasi Z., Laszlo V., Renyi-Vamos F., Galffy G., Bodor C., Dome B, & Moldvay J. (2021). EGFR variant allele frequency predicts EGFR-TKI efficacy in lung adenocarcinoma: a multicenter study. Translational Lung Cancer Research, 10(2), 662-674.

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FFPE DNA Extraction and EGFR Mutation Detection

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Genomic DNA was isolated from biopsy tissue sections using a standard protocol. In particular, paraffin was removed from formalin-fixed paraffin-embedded (FFPE) samples with Bio-Clear (Bio-optica, Milan, Italy), and DNA was purified using the QIAamp DNA FFPE Tissue kit (Qiagen, Valencia, CA, USA). DNA quantitation and quality assessment were carried out with both a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and Qubit^® 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). DNA fragmentation status was evaluated with an Agilent 2200 TapeStation system using Genomic DNA ScreenTape assay (Agilent Technologies, Santa Clara, CA, USA) able to produce a DNA integrity number (DIN). Quantitative measurements of EGFR mutations were performed using the Therascreen™ EGFR Pyro Kit (Qiagen), for the detection of mutations in codon 719 (exon 18), in codons 768 and 790 (exon 20), and in codons 858 to 861 (exon 21), along with deletions and complex mutations in exon 19 of the human EGFR gene on genomic DNA. Each pyrosequencing assay was performed on the PyroMark Q24 system (Qiagen), following the manufacturer’s instructions.

Casula M., Pisano M., Paliogiannis P., Colombino M., Sini M.C., Zinellu A., Santeufemia D., Manca A., Casula S., Tore S., Lobrano R., Cossu A, & Palmieri G. (2023). Comparison between Three Different Techniques for the Detection of EGFR Mutations in Liquid Biopsies of Patients with Advanced Stage Lung Adenocarcinoma. International Journal of Molecular Sciences, 24(7), 6410.

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EGFR Mutation Detection in FFPE Samples

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Formalin-fixed and paraffin-embedded tissues genomic DNA was extracted from 3–6 (10 μm thick) sequential sections through QIAamp DNA FFPE tissue kit (Qiagen) according to the manufacturer’s instructions and checked for adequacy by NanoDrop. Primers used were previously developed in our lab and each sample was analyzed using both our primers and the Therascreen EGFR Pyro Kit primers (Qiagen) for validation. In brief, when using our own developed primers, PCR reactions were performed on 100 ng of DNA in a total volume of 50 μL containing 5× buffer, 200 μM dNTP, 0.5 μM of each primer, and 0.2U Taq polymerase (Takara), with the following cycling conditions: 95 °C for 5 min followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 56/60 °C for 30 s, extension at 72 °C for 30 s. A final extension at 72 °C for 5 min was finally performed. PCR of the Therascreen EGFR Pyro Kit were performed according to the manufacturer directives (Qiagen). Amplicons were analyzed by gel electrophoresis on a 2% agarose gel stained with ethidium bromide and visualized by ultraviolet trans-illumination.

Abdelmaksoud-Dammak R., Ammous-Boukhris N., Saadallah-Kallel A., Charfi S., Khemiri S., Khemakhem R., Kallel N., Ben Kridis-Rejeb W., Sallemi-Boudawara T., Khanfir A., Yangui I., Daoud J, & Mokdad-Gargouri R. (2022). Predominance of the Rare EGFR Mutation p.L861Q in Tunisian Patients with Non-Small Cell Lung Carcinoma. Genes, 13(8), 1499.

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EGFR Mutation Detection Protocols

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The following reference methods were used for routine analysis of the EGFR mutational status: NGS using the Ion Torrent AmpliSeq Colon and Lung Cancer Research Panel version 2 (Thermo Fisher Scientific, Waltham, MA; n Z 12 samples), the Oncomine Focus Assay (Thermo Fisher Scientific; n Z 25), the HaloPlex Cancer Research Panel (Agilent Technologies, Santa Clara, CA; n Z 20), or inhouseedeveloped NGS (n Z 3); pyrosequencing using the therascreen EGFR Pyro kit (Qiagen, Venlo, the Netherlands; n Z 6) or in-houseedeveloped pyrosequencing (n Z 34); PCR-based assays using the cobas EGFR Mutation Test version 2 (Roche Molecular Systems, Pleasanton, CA; n Z 130) or the therascreen EGFR RGQ PCR Kit version 1 or 2 (Qiagen; n Z 71); mass spectrometric methods using the MassArray Lung Cancer Panel (Diatech, Jesi, Italy; n Z 30); and Sanger sequencing using in-house methods (n Z 118). Commercial assays were performed according to the manufacturer's instructions. Analytical sensitivities for the commercial assays can be found in the respective manufacturer's instructions. In-house methods were performed by adopting different protocols and equipment at each site.

Evrard S.M., Taranchon-Clermont E., Rouquette I., Murray S., Dintner S., Nam-Apostolopoulos Y.C., Bellosillo B., Varela-Rodriguez M., Nadal E., Wiedorn K.H., Melchior L., Andrew E., Jones M., Ridgway J., Frykman C., Lind L., Rot M., Kern I., Speel E.J.M., Roemen G.M.J.M., Trincheri N., Freiberger S.N, & Rechsteiner M. (2019). Multicenter Evaluation of the Fully Automated PCR-Based Idylla EGFR Mutation Assay on Formalin-Fixed, Paraffin-Embedded Tissue of Human Lung Cancer. The Journal of molecular diagnostics : JMD, 21(6).

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