We employed our ISO 15189:2012 certified orthogonal diagnostic tests for solid tumors to check and confirm the NGS data. These include the Therascreen EGFR RGQ PCR kit (Qiagen) that detects 29 somatic mutations in exons 18 to 21 of EGFR, and the Therascreen BRAF RGQ PCR kit (Qiagen) for analysis of five somatic mutations at amino acid V600 of BRAF. For RAS screening, we used an in-house developed KRAS G12-G13 qPCR to screen for 7 KRAS mutations in codons 12 and 13, complemented with pyrosequencing on the PyroMark Q24 (Qiagen) with the Therascreen RAS extension Pyro kit (Qiagen) for detection of mutations in KRAS codons 59, 61, 117 and 146. With the same method, mutations in codons 12, 13, 59, 61, 117 and 146 of NRAS were screened for by the Therascreen NRAS Pyro kit (Qiagen).
Therascreen nras pyro kit
Manufactured by Qiagen
Sourced in Germany
The Therascreen NRAS Pyro Kit is a laboratory equipment product developed by Qiagen. It is designed for the detection and analysis of NRAS mutations in cancer samples using pyrosequencing technology. The kit provides a standardized and reliable solution for the genetic analysis of NRAS gene status.
Automatically generated - may contain errors
Lab products found in correlation
Pyromark q24, by Qiagen (3 mentions)
Therascreen ras extension pyro kit, by Qiagen (3 mentions)
Therascreen braf pyro kit, by Qiagen (3 mentions)
Therascreen braf rgq pcr kit, by Qiagen (1 mentions)
Therascreen egfr rgq pcr kit, by Qiagen (1 mentions)
Therascreen kras pyro kit, by Qiagen (1 mentions)
Pyromark q24 system, by Qiagen (1 mentions)
Pyromark q24 vacuum workstation, by Qiagen (1 mentions)
Qiamp dna ffpe tissue kit, by Qiagen (1 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (1 mentions)
6 protocols using therascreen nras pyro kit
1
Comprehensive Orthogonal Genomic Profiling
2
Molecular Profiling for Metastatic Colorectal Cancer
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During screening, CTCs were determined by central testing (San Carlos Hospital, Madrid, Spain). Peripheral blood (10 ml) was collected in CellSave® Preservative Tubes (Veridex LLC, Raritan, NJ), and CTCs were enumerated using the CellSearch® Tumor Circulating Cell kit (Veridex LLC, Raritan, NJ). Mutational analyses of KRAS, NRAS, BRAF and PIK3CA genes were done on primary tumour or metastatic tissue samples at six reference laboratories. Mutations in KRAS exons 2 and 3, BRAFV600 and PIK3CA exons 9 and 20 were determined by the Cobas® test (Roche Diagnostics, Indianapolis, IN) and mutations in KRAS exon 4 and NRAS exons 2, 3 and 4 were analysed by pyrosequencing [Therascreen® NRAS Pyro Kit or Therascreen® RAS Extension Pyro Kit (Qiagen, Venlo, Netherlands)].
Tumour assessments were carried out using computed tomography of the chest, abdomen and pelvic region at baseline and then every 12 weeks until disease progression, and evaluated according to RECIST, version 1.1. After discontinuing treatment, patients were followed every 3 months for 2 years for any tumour assessments and survival. Toxicities were graded according to the National Cancer Institute Common Toxicity Criteria for Adverse Events, version 4.0.
Tumour assessments were carried out using computed tomography of the chest, abdomen and pelvic region at baseline and then every 12 weeks until disease progression, and evaluated according to RECIST, version 1.1. After discontinuing treatment, patients were followed every 3 months for 2 years for any tumour assessments and survival. Toxicities were graded according to the National Cancer Institute Common Toxicity Criteria for Adverse Events, version 4.0.
3
Molecular Profiling of Primary and Metastatic Tumors
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All primary tumors were analyzed for KRAS exon 2 mutations. For metastases, molecular analyses of KRAS, NRAS and BRAF were carried out by using the Qiagen therascreen kits (therascreen BRAF Pyro Kit, therascreen RAS Extension Pyro Kit, therascreen KRAS Pyro Kit, therascreen NRAS Pyro Kit) as previously described [49 (link)]. PIK3CA was analyzed by applying high resolution melting analysis (HRM), mutations were confirmed by Sanger sequencing as described beforehand [50 (link)]. Mismatch repair deficiency/microsatellite instability (MSI) was evaluated by means of immunohistochemistry following the locally established protocol with the ready to use antibodies MLH1 (Clon ES05), MSH2 (Clon FE11), MSH6 (Clon Epi 49) and PMS2 (Clon EP51) (Dako, Agilent technologies, Glostrup, Denmark).
4
NRAS Mutational Analysis by Pyrosequencing
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NRAS mutation status of IPC-298 cells was determined by DNA pyrosequencing on the PyroMark Q24 System (Qiagen, Hilden, Germany) following the manufacturer's instructions. After lysing cells in standard lysis buffer, creation of single-stranded DNA was conducted using the PyroMark Q24 Vacuum Workstation; the therascreen NRAS Pyro Kit (Qiagen, Hilden, Germany) was used to perform pyrosequencing according to the manufacturer's protocol. Sequence data was analyzed using the PyroMark Q24 Software (v. 2.0, Qiagen, Hilden, Germany).
5
BRAF and NRAS Mutation Analysis by Pyrosequencing
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DNA extraction was performed with the QiAmp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) and pyrosequencing analysis was performed as previously described with the therascreen BRAF Pyro Kit (Qiagen) and the therascreen NRAS Pyro Kit (Qiagen) [47 (link)–49 (link)]. The platform of the LCEP, (Nice, France) has been accredited for molecular testing by pyrosequencing of exons 11 and 15 of the BRAF gene and for codon 12, 13 and 61 of the NRAS gene. The laboratory holds full COFRAC (“Comité Français d'Accréditation”) accreditation for these assays, according to the ISO 15189 norm (http://www.cofrac.fr ). The analytical sensitivity of the pyrosequencing method was considered to be 5% according to the manufacturer (Qiagen). Pyrogram profile analyses and interpretation of the results were done blindly with PyromarkQ24 (Qiagen) by five independent assessors (EL, MI, VL, VH OB).
6
Mutational Analysis of BRAF and NRAS
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The BRAF and NRAS mutational status was determined for 30/65 (46%) cases. Tumour DNA was isolated from FFPE tissue samples using the QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Pyrosequencing of NRAS exon 1 (codon 12 and 13) and exon 2 (codons 60 and 61) using the Therascreen NRAS Pyro kit (Qiagen, Hilden, Germany) and BRAF exon 15 using the Therascreen BRAF Pyro Kit (Qiagen, Hilden, Germany), was performed as previously described
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