For each sample, codons 12, 13, and 61 of the KRAS Gene, and codon V600E of the BRAF Gene were screened by Pyrosequencing and Competitive Allele-Specific TaqMan PCR (Cast-PCR), respectively. Pyrosequencing of KRAS codons 12, 13, and 61 was performed using the Therascreen KRAS Pyro Kit (QIAGEN) by manufacture's protocols. For pyrosequencing preparation, KRAS was first amplified by primers, one of which was biotinylated to immobilize with streptavidin beads (GE healthcare). PCR-pyrosequencing reaction was carried out on thermocycler (Eppendorf), containing 10 ng of genomic DNA. Two sets of the seq primer (Therascreen KRAS Pyro Kit QIAGEN) were used to analyze mutations in codons 12, 13, and 61. Pyromark Q24 version 2 software (QIAGEN, Hilden, Germany) was applied to analyze pyrosequencing results. Detection limit for KRAS mutations was obtained as 3% according to the Therascreen KRAS Pyro Kit (QIAGEN) kit. Cast-PCR™ was performed in 20-μl reactions comprising 50 ng DNA template, 1X Genotyping Master Mix (Applied Biosystems), and 1X of TaqMan Mutation detection assays. PCR was run on a real-time PCR (7500 ABI). Cycling conditions were as follows: one cycle consisting 95°C for 10 min, followed by 5 cycles of 92°C for 15 s, 58°C for 1 min, 45 cycles, 92°C for 15 s, and 60°C for 1 min.
Therascreen kras pyro kit
Manufactured by Qiagen
Sourced in Germany
The Therascreen KRAS Pyro Kit is a laboratory equipment product developed by Qiagen. It is designed to detect mutations in the KRAS gene, which is commonly associated with various types of cancer. The kit utilizes pyrosequencing technology to analyze DNA samples and identify specific KRAS gene variations.
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Lab products found in correlation
Therascreen ras extension pyro kit, by Qiagen (6 mentions)
Pyromark q24 system, by Qiagen (4 mentions)
Qiaamp minelute spin columns, by Qiagen (3 mentions)
Therascreen braf pyro kit, by Qiagen (3 mentions)
Veriti 96 well fast thermal cycler, by Thermo Fisher Scientific (2 mentions)
Pyromark assay design 2, by Qiagen (2 mentions)
Pyromark q24, by Qiagen (2 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (2 mentions)
Pyromark pcr master mix 2x, by Qiagen (2 mentions)
Coralload concentrate 10x, by Qiagen (2 mentions)
Streptavidin sepharose high performance beads, by Qiagen (2 mentions)
Pyromark q24 plate, by Qiagen (2 mentions)
Genotyping master mix, by Thermo Fisher Scientific (1 mentions)
Thermocycler, by Eppendorf (1 mentions)
Streptavidin beads, by GE Healthcare (1 mentions)
T100 thermocycler, by Bio-Rad (1 mentions)
Pyromark q24 platform, by Qiagen (1 mentions)
Ion ampliseq colon and lung cancer panel v2, by Thermo Fisher Scientific (1 mentions)
Therascreen nras pyro kit, by Qiagen (1 mentions)
Streptavidin sepharose high performance, by GE Healthcare (1 mentions)
16 protocols using therascreen kras pyro kit
1
KRAS and BRAF Mutation Analysis
2
KRAS Codon 12/13 Pyrosequencing
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Pyrosequencing of the KRAS codon 12/13 region was performed using the Therascreen KRAS Pyro Kit (Qiagen) as recommended by the manufacturer. 2 ng of DNA were used per analysis. PCR amplification of the target region was performed on a T-100 thermocycler (Biorad). For the pyrosequencing reaction on the PyroMark Q24 platform (Qiagen), amplicons were immobilized to the wells of a PyroMark Q24 plate using streptavidin high performance beads (GE Healthcare). Pyrosequencing results were analysed using the PyroMark Q24 software version 2.0 with the Therascreen KRAS Pyro-plugin report, which already incorporated the thresholds for mutation calls (detection limit for the mutation (LOD) + 3 %).
3
Pyrosequencing Analysis of KRAS and BRAF
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The PyroMark Q24 system (Qiagen GmbH, Hilden, Germany) was used for pyrosequencing analysis of KRAS and BRAF mutations in DNA from 1 mm formalin-fixed, paraffin-embedded or fresh frozen tumour tissue cores taken from areas with >90 % tumour cells, as previously described [35 (link)]. In brief, DNA was isolated from tumour tissue using QIAamp MinElute spin columns (Qiagen) and DNA regions of interest were PCR-amplified (Veriti 96-Well Fast Thermal Cycler, Applied Biosystems Inc., Foster City, CA, USA).
Detection of mutations in KRAS codons 12 and 13 was performed using Therascreen KRAS Pyro Kit (Qiagen). Analysis of BRAF mutation hotspots in codons 600 and 601 was performed using previously published PCR primers (Richman, JCO 2009) and a novel BRAF sequencing primer (5′-TGATTTTGGTCTAGCTACA-3′) which was designed using the PyroMark Assay Design 2.0 software (Qiagen). All samples with a potential low-level mutation were re-analysed.
Detection of mutations in KRAS codons 12 and 13 was performed using Therascreen KRAS Pyro Kit (Qiagen). Analysis of BRAF mutation hotspots in codons 600 and 601 was performed using previously published PCR primers (Richman, JCO 2009) and a novel BRAF sequencing primer (5′-TGATTTTGGTCTAGCTACA-3′) which was designed using the PyroMark Assay Design 2.0 software (Qiagen). All samples with a potential low-level mutation were re-analysed.
4
Molecular Profiling of Tumor Samples
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Tumor tissue was available for all patients and the KRAS mutation status was assessed from the primary tumor (all patients except DR4) or metastatic sites (DR4). In addition, the primary tumors from patient DR7, DR9, and DR13 were examined for changes in TP53 and/or BRAF. The Ion AmpliSeq Colon and Lung Cancer Panel v2 (Thermo Fisher Scientific), the Therascreen KRAS Pyro Kit (QIAGEN) or the Idylla KRAS and NRAS-BRAF-EGFR S492R Mutation Assays (Biocartis) were used to establish the mutation status in these genes directly from formalin-fixed paraffin-embedded (FFPE) tissue sections. Tumor genotyping was carried out at the Diagnostic and Research Institute of Pathology, Medical University of Graz and at the Department of Pathology, General Hospital Graz II, Graz, Austria.
For 9 of 11 patients (82%), specific mutations were available from the primary tissue, in one patient (DR4) only metastatic material was available for molecular profiling and in the tumor of one patient (DR13), no mutation was identified.
For 9 of 11 patients (82%), specific mutations were available from the primary tissue, in one patient (DR4) only metastatic material was available for molecular profiling and in the tumor of one patient (DR13), no mutation was identified.
5
KRAS, NRAS, and BRAF Mutation Analysis
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DNA was extracted from three 10 μm-slides from FFPE tumor tissue samples using the QIAamp DNA FFPE Tissue Kit (QIAGEN, Milan, Italy), following the manufacturer’s protocol. In order to minimize contamination by normal cells, the tumor areas dissected for DNA extraction contained at least 70% of tumor cells. KRAS, NRAS and BRAF mutational analyses were carried out by pyrosequencing on chemo-naïve primary or metastatic samples, as previously described [26 (link)]. RAS and BRAF mutational analysis were performed using the therascreen KRAS Pyro Kit, therascreen RAS Extension Pyro Kit and therascreen BRAF Pyro Kit (Qiagen, Milan, Italy). Mutation status was determined by pyrosequencing on the Qiagen PyroMark Q24.
6
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).
7
Quantitative KRAS Mutation Analysis
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Quantitative analysis of KRAS mutations was performed by pyrosequencing in accordance with routine practice at the Department of Pathology at Fundación Jiménez Díaz University Hospital, using the CE-IVD marked Therascreen KRAS Pyro kit (Qiagen), according to the manufacturer’s protocols. Each PCR product was analyzed by pyrosequencing using the Therascreen KRAS Pyro reagents (Qiagen), Streptavidin Sepharose High Performance (GE Healthcare Bio-Science AB, Uppsala, Sweden), and a PyroMark Q24 instrument (Qiagen).
8
Comparative Analysis of KRAS Mutation Testing
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At several centers to guide cancer therapy, the KRAS mutational status had been assessed previously on FFPE material using commercial routine tests.
The cobas® KRAS Mutation Test (Roche), the Ion Torrent AmpliSeq™ Colon and Lung Cancer Research Panel (Thermo Fisher Scientific), therascreen® KRAS Pyro Kit (Qiagen), therascreen® RAS Extension Pyro Kit (Qiagen), the KRAS, BRAF, PIK3CA* Array (Randox Molecular, Crumlin, UK), and the QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4) (DiaCarta, Hayward, CA), were performed according to the manufacturer’s instructions.
The cobas® KRAS Mutation Test (Roche), the Ion Torrent AmpliSeq™ Colon and Lung Cancer Research Panel (Thermo Fisher Scientific), therascreen® KRAS Pyro Kit (Qiagen), therascreen® RAS Extension Pyro Kit (Qiagen), the KRAS, BRAF, PIK3CA* Array (Randox Molecular, Crumlin, UK), and the QClamp™ KRAS Codon Specific Mutation Detection Kit (Exon 2, 3, 4) (DiaCarta, Hayward, CA), were performed according to the manufacturer’s instructions.
9
Analysis of EGFR and KRAS Mutations
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The PyroMark Q24 system (QIAGEN) was used for pyrosequencing analysis of EGFR and KRAS mutations in DNA from 1 mm formalin-fixed, paraffin-embedded tumour tissue cores. In brief, genomic DNA was extracted from tumour tissue in QIAamp MinElute spin columns (QIAGEN) and DNA regions of interest were amplified by PCR (Veriti 96 Well Fast Thermal Cykler, Applied Biosystems Inc., Foster City CA). Using therascreen KRAS Pyro Kit and EGFR Pyro Kit (QIAGEN) KRAS mutations of codon 12, 13 and 61 and EGFR deletions in exon 19 and mutations in exon 18 codon 719, exon 20 codon 768 plus 790 and exon 21 codon 858–861 were analysed. All samples with a potential low-level mutation were reexamined in duplicates.
10
RAS Mutation Detection using Pyrosequencing
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The TheraScreen® KRAS Pyro kit (for KRAS codons 12 and 13), and the TheraScreen® RAS Extension Pyro kit (for KRAS codons 59/61, 117 and 146, and NRAS codons 12, 13, 59, 61, 117 and 146) (Qiagen, GERMANY), were used for RAS mutations testing, according to the manufacturer’s instructions. Briefly, 5 μl of template DNA (2–10 ng of genomic DNA) were amplified by polymerase chain reaction (PCR) in a 20 μl volume containing 12.5 μl of PyroMark® PCR Master Mix 2x, 2.5 μl of Coral Load Concentrate 10x, 4 μl of nuclease-free water, and 1 μl of the corresponding set of PCR primers (Qiagen). 10 μl of PCR products were immobilized to Streptavidin Sepharose High Performance beads (Qiagen) to prepare the single‑stranded DNA. The corresponding sequencing primers were allowed to anneal to the DNA using a PyroMark Q24 plate and a vacuum workstation (Qiagen). The sequences were analyzed using Pyromark Q24 software in the AQ analysis mode. In each run, a negative control (without template DNA) and an unmethylated control DNA, provided by the kit as a positive control for PCR and sequencing reactions were included.
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