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point mutation detection  (Qiagen)


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    Qiagen point mutation detection
    Point Mutation Detection, supplied by Qiagen, used in various techniques. Bioz Stars score: 93/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mutation+detection+pcr+kit/Type-it+Mutation+Detect+PCR+Kit/us12592321-783-4-10
    Average 93 stars, based on 57 article reviews
    point mutation detection - by Bioz Stars, 2026-10
    93/100 stars

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    Related Articles

    Polymerase Chain Reaction:

    Article Title: Predictors of Enteric Pathogens in the Domestic Environment from Human and Animal Sources in Rural Bangladesh
    Article Snippet: .. PCR was performed using the Type-It Mutation Detection PCR Kit (Qiagen, Germantown, MD). ..

    Article Title: [2401.06169] Deep Learning model predicts the c-Kit-11 mutational status of canine cutaneous mast cell tumors by HE stained histological slides
    Article Snippet: .. PCR-based identification of c-Kit exon 11 Mutational status Neoplastic cells were isolated from FFPE MCTs with laser-capture microdissection (LCM) as previously described.24 The exon 11 of the c-Kit gene was amplified by PCR using the primer pair at the 5’ end of the exon 11 (PE1: 5’- CCATGTATGAAGTACAGTGGAAG-3’ sense, bp 1,657–1,680 of exon 11) and the 5’- end of intron 11 (PE2: 5’GTTCCCTAAAGTCATTGTTACACG-3’ anti-sense, nucleotides 43–66 of intron 11).3 A Mutation Detection PCR Kit (Qiagen) was used for the amplification.2 PCRs were prepared in a 25-μL total reaction volume, with 10 to 25 ng of LCM-extracted DNA and 5 pmol of each primer 0.5 U of Taq polymerase (Invitrogen, Carlsbad, CA), and final concentrations of 80 μM deoxynucleoside triphosphate, 2 mM MgCl2, 20 mM Tris–HCl, and 50 μl of KCl.24 Cycling conditions were as follows: 95 °C for 5 minutes; 35 cycles at 95 °C for 30 seconds, 58 °C for 90 seconds, 72 °C for 30 seconds; followed by 68 °C for 10 minutes.2 PCR products were visualized on the QIAxcel Capillary Electrophoresis System (Qiagen).2 .. To investigate the dependency of the mutational status prediction on the staining protocol, we applied two stains (Stain A, Stain B) to the same tissue section, which was de-stained in between both staining steps, using the following protocol: HE-stain A – Paraffin sections of all tumors were originally stained at MSU Veterinary Diagnostic Laboratory using the VENTANA HE600 system from Roche.

    Article Title: Artificial intelligence can be trained to predict c-KIT -11 mutational status of canine mast cell tumors from hematoxylin and eosin-stained histological slides
    Article Snippet: .. A Mutation Detection PCR Kit (Qiagen) was used for the amplification. .. PCRs were prepared in a 25 μl total reaction volume, with 10 to 25 ng of extracted DNA; 5 pmol of each primer; 0.5 U of Taq polymerase (Invitrogen, Carlsbad, California); and final concentrations of 80 μm deoxynucleoside triphosphate, 2 mM MgCl 2 , 20 mM Tris-HCl, and 50 μl of KCl.

    Article Title: Internal Tandem Duplication of Exon 8 of c-kit Is Associated With Longer Total Survival in Canine Cutaneous Mast Cell Tumors.
    Article Snippet: Canine cutaneous mast cell tumors (ccMCTs) have a highly variable biological behavior and accurate prognostication is essential for therapeutic intervention.. Internal tandem duplications (ITD) of exon 11 are the most commonly detected c-kit mutation in ccMCTs and are associated with poor prognosis and increased cellular proliferation.. The prognostic value of detecting mutations in other exons of c-kit has not been systematically examined.

    Article Title: Mutations in Exons 8 and 11 of c-kit Gene in Canine Subcutaneous Mast Cell Tumors and Their Association with Cell Proliferation
    Article Snippet: .. Amplifications for both primer sets were performed using the Type-it Mutation Detection PCR Kit (Qiagen) as previously described [ , ]. .. PCR products were visualized on the QIAxcel Capillary Electrophoresis System (Qiagen) [ ].

    Article Title: Artificial intelligence can be trained to predict c-KIT -11 mutational status of canine mast cell tumors from hematoxylin and eosin-stained histological slides.
    Article Snippet: .. PCR-Based Identification of ITD Mutation in the c-KIT Exon 11 The PCR-based identification of c-KIT-11-ITD was done at the Michigan State University as part of the MCT panel for routine diagnostics, as previously described.32 Exon 11 of the c-KIT gene was amplified by PCR using the primer pair at the 5’ end of the exon 11 (PE1: 5’-CCATGTATGAAGTACAGTGGAAG-3’ sense, bp 1657–1680 of exon 11) and the 5’ end of intron 11 (PE2: 5’GTTCCCTAAAGTCATTGTTACACG-3’ anti-sense, nucleotides 43–66 of intron 11).4 A Mutation Detection PCR Kit (Qiagen) was used for the amplification.3 PCRs were prepared in a 25 μl total reaction volume, with 10 to 25 ng of extracted DNA; 5 pmol of each primer; 0.5 U of Taq polymerase (Invitrogen, Carlsbad, California); and final concentrations of 80 μm deoxynucleoside triphosphate, 2 mM MgCl2, 20 mM Tris-HCl, and 50 μl of KCl.32 Cycling conditions were as follows: 95°C for 5 minutes; 35 cycles at 95°C for 30 seconds, 58°C for 90 seconds, 72°C for 30 seconds; followed by 68°C for 10 minutes.3 PCR products were visualized on the QIAxcel Capillary Electrophoresis System (Qiagen).3 Staining Variants To investigate the dependency of the mutational status prediction on the staining protocol, we applied 2 stains (stains A and B) to the same tissue slice, which was de-stained in-between the staining steps, using the following protocol: HE—stain A—Paraffin sections of all tumors were originally stained at Michigan State University Veterinary Diagnostic Laboratory using the Ventana HE600 system from Roche. .. Dry deparaffinization, rehydration, and HE staining were run according to a standard protocol11 and using Ventana HE 600 Hematoxylin (Roche, 07024282001) and Ventana HE 600 Eosin (Roche, 06544304001).

    Article Title: Development of a RACE-based RNA-Seq approach to characterize the T-cell receptor repertoire of porcine γδ T cells.
    Article Snippet: Recent data suggest that porcine γδ T cells exhibit a similar degree of functional plasticity as human and murine γδ T cells.. Due to the high frequency of TCR-γδ cells in blood and secondary lymphatic organs, the pig is an attractive model to study these cells, especially their combined features of the innate and the adaptive immune system.. Using a 5’ RACE-like approach, we translated a human/murine NGS library preparation strategy to capture full-length V-(D)-J TRG and TRD clonotypes in swine.

    Mutagenesis:

    Article Title: Predictors of Enteric Pathogens in the Domestic Environment from Human and Animal Sources in Rural Bangladesh
    Article Snippet: .. PCR was performed using the Type-It Mutation Detection PCR Kit (Qiagen, Germantown, MD). ..

    Article Title: [2401.06169] Deep Learning model predicts the c-Kit-11 mutational status of canine cutaneous mast cell tumors by HE stained histological slides
    Article Snippet: .. PCR-based identification of c-Kit exon 11 Mutational status Neoplastic cells were isolated from FFPE MCTs with laser-capture microdissection (LCM) as previously described.24 The exon 11 of the c-Kit gene was amplified by PCR using the primer pair at the 5’ end of the exon 11 (PE1: 5’- CCATGTATGAAGTACAGTGGAAG-3’ sense, bp 1,657–1,680 of exon 11) and the 5’- end of intron 11 (PE2: 5’GTTCCCTAAAGTCATTGTTACACG-3’ anti-sense, nucleotides 43–66 of intron 11).3 A Mutation Detection PCR Kit (Qiagen) was used for the amplification.2 PCRs were prepared in a 25-μL total reaction volume, with 10 to 25 ng of LCM-extracted DNA and 5 pmol of each primer 0.5 U of Taq polymerase (Invitrogen, Carlsbad, CA), and final concentrations of 80 μM deoxynucleoside triphosphate, 2 mM MgCl2, 20 mM Tris–HCl, and 50 μl of KCl.24 Cycling conditions were as follows: 95 °C for 5 minutes; 35 cycles at 95 °C for 30 seconds, 58 °C for 90 seconds, 72 °C for 30 seconds; followed by 68 °C for 10 minutes.2 PCR products were visualized on the QIAxcel Capillary Electrophoresis System (Qiagen).2 .. To investigate the dependency of the mutational status prediction on the staining protocol, we applied two stains (Stain A, Stain B) to the same tissue section, which was de-stained in between both staining steps, using the following protocol: HE-stain A – Paraffin sections of all tumors were originally stained at MSU Veterinary Diagnostic Laboratory using the VENTANA HE600 system from Roche.

    Article Title: Artificial intelligence can be trained to predict c-KIT -11 mutational status of canine mast cell tumors from hematoxylin and eosin-stained histological slides
    Article Snippet: .. A Mutation Detection PCR Kit (Qiagen) was used for the amplification. .. PCRs were prepared in a 25 μl total reaction volume, with 10 to 25 ng of extracted DNA; 5 pmol of each primer; 0.5 U of Taq polymerase (Invitrogen, Carlsbad, California); and final concentrations of 80 μm deoxynucleoside triphosphate, 2 mM MgCl 2 , 20 mM Tris-HCl, and 50 μl of KCl.

    Article Title: Internal Tandem Duplication of Exon 8 of c-kit Is Associated With Longer Total Survival in Canine Cutaneous Mast Cell Tumors.
    Article Snippet: Canine cutaneous mast cell tumors (ccMCTs) have a highly variable biological behavior and accurate prognostication is essential for therapeutic intervention.. Internal tandem duplications (ITD) of exon 11 are the most commonly detected c-kit mutation in ccMCTs and are associated with poor prognosis and increased cellular proliferation.. The prognostic value of detecting mutations in other exons of c-kit has not been systematically examined.

    Article Title: Mutations in Exons 8 and 11 of c-kit Gene in Canine Subcutaneous Mast Cell Tumors and Their Association with Cell Proliferation
    Article Snippet: .. Amplifications for both primer sets were performed using the Type-it Mutation Detection PCR Kit (Qiagen) as previously described [ , ]. .. PCR products were visualized on the QIAxcel Capillary Electrophoresis System (Qiagen) [ ].

    Article Title: Artificial intelligence can be trained to predict c-KIT -11 mutational status of canine mast cell tumors from hematoxylin and eosin-stained histological slides.
    Article Snippet: .. PCR-Based Identification of ITD Mutation in the c-KIT Exon 11 The PCR-based identification of c-KIT-11-ITD was done at the Michigan State University as part of the MCT panel for routine diagnostics, as previously described.32 Exon 11 of the c-KIT gene was amplified by PCR using the primer pair at the 5’ end of the exon 11 (PE1: 5’-CCATGTATGAAGTACAGTGGAAG-3’ sense, bp 1657–1680 of exon 11) and the 5’ end of intron 11 (PE2: 5’GTTCCCTAAAGTCATTGTTACACG-3’ anti-sense, nucleotides 43–66 of intron 11).4 A Mutation Detection PCR Kit (Qiagen) was used for the amplification.3 PCRs were prepared in a 25 μl total reaction volume, with 10 to 25 ng of extracted DNA; 5 pmol of each primer; 0.5 U of Taq polymerase (Invitrogen, Carlsbad, California); and final concentrations of 80 μm deoxynucleoside triphosphate, 2 mM MgCl2, 20 mM Tris-HCl, and 50 μl of KCl.32 Cycling conditions were as follows: 95°C for 5 minutes; 35 cycles at 95°C for 30 seconds, 58°C for 90 seconds, 72°C for 30 seconds; followed by 68°C for 10 minutes.3 PCR products were visualized on the QIAxcel Capillary Electrophoresis System (Qiagen).3 Staining Variants To investigate the dependency of the mutational status prediction on the staining protocol, we applied 2 stains (stains A and B) to the same tissue slice, which was de-stained in-between the staining steps, using the following protocol: HE—stain A—Paraffin sections of all tumors were originally stained at Michigan State University Veterinary Diagnostic Laboratory using the Ventana HE600 system from Roche. .. Dry deparaffinization, rehydration, and HE staining were run according to a standard protocol11 and using Ventana HE 600 Hematoxylin (Roche, 07024282001) and Ventana HE 600 Eosin (Roche, 06544304001).

    Article Title: Development of a RACE-based RNA-Seq approach to characterize the T-cell receptor repertoire of porcine γδ T cells.
    Article Snippet: Recent data suggest that porcine γδ T cells exhibit a similar degree of functional plasticity as human and murine γδ T cells.. Due to the high frequency of TCR-γδ cells in blood and secondary lymphatic organs, the pig is an attractive model to study these cells, especially their combined features of the innate and the adaptive immune system.. Using a 5’ RACE-like approach, we translated a human/murine NGS library preparation strategy to capture full-length V-(D)-J TRG and TRD clonotypes in swine.

    Isolation:

    Article Title: [2401.06169] Deep Learning model predicts the c-Kit-11 mutational status of canine cutaneous mast cell tumors by HE stained histological slides
    Article Snippet: .. PCR-based identification of c-Kit exon 11 Mutational status Neoplastic cells were isolated from FFPE MCTs with laser-capture microdissection (LCM) as previously described.24 The exon 11 of the c-Kit gene was amplified by PCR using the primer pair at the 5’ end of the exon 11 (PE1: 5’- CCATGTATGAAGTACAGTGGAAG-3’ sense, bp 1,657–1,680 of exon 11) and the 5’- end of intron 11 (PE2: 5’GTTCCCTAAAGTCATTGTTACACG-3’ anti-sense, nucleotides 43–66 of intron 11).3 A Mutation Detection PCR Kit (Qiagen) was used for the amplification.2 PCRs were prepared in a 25-μL total reaction volume, with 10 to 25 ng of LCM-extracted DNA and 5 pmol of each primer 0.5 U of Taq polymerase (Invitrogen, Carlsbad, CA), and final concentrations of 80 μM deoxynucleoside triphosphate, 2 mM MgCl2, 20 mM Tris–HCl, and 50 μl of KCl.24 Cycling conditions were as follows: 95 °C for 5 minutes; 35 cycles at 95 °C for 30 seconds, 58 °C for 90 seconds, 72 °C for 30 seconds; followed by 68 °C for 10 minutes.2 PCR products were visualized on the QIAxcel Capillary Electrophoresis System (Qiagen).2 .. To investigate the dependency of the mutational status prediction on the staining protocol, we applied two stains (Stain A, Stain B) to the same tissue section, which was de-stained in between both staining steps, using the following protocol: HE-stain A – Paraffin sections of all tumors were originally stained at MSU Veterinary Diagnostic Laboratory using the VENTANA HE600 system from Roche.

    Formalin-fixed Paraffin-Embedded:

    Article Title: [2401.06169] Deep Learning model predicts the c-Kit-11 mutational status of canine cutaneous mast cell tumors by HE stained histological slides
    Article Snippet: .. PCR-based identification of c-Kit exon 11 Mutational status Neoplastic cells were isolated from FFPE MCTs with laser-capture microdissection (LCM) as previously described.24 The exon 11 of the c-Kit gene was amplified by PCR using the primer pair at the 5’ end of the exon 11 (PE1: 5’- CCATGTATGAAGTACAGTGGAAG-3’ sense, bp 1,657–1,680 of exon 11) and the 5’- end of intron 11 (PE2: 5’GTTCCCTAAAGTCATTGTTACACG-3’ anti-sense, nucleotides 43–66 of intron 11).3 A Mutation Detection PCR Kit (Qiagen) was used for the amplification.2 PCRs were prepared in a 25-μL total reaction volume, with 10 to 25 ng of LCM-extracted DNA and 5 pmol of each primer 0.5 U of Taq polymerase (Invitrogen, Carlsbad, CA), and final concentrations of 80 μM deoxynucleoside triphosphate, 2 mM MgCl2, 20 mM Tris–HCl, and 50 μl of KCl.24 Cycling conditions were as follows: 95 °C for 5 minutes; 35 cycles at 95 °C for 30 seconds, 58 °C for 90 seconds, 72 °C for 30 seconds; followed by 68 °C for 10 minutes.2 PCR products were visualized on the QIAxcel Capillary Electrophoresis System (Qiagen).2 .. To investigate the dependency of the mutational status prediction on the staining protocol, we applied two stains (Stain A, Stain B) to the same tissue section, which was de-stained in between both staining steps, using the following protocol: HE-stain A – Paraffin sections of all tumors were originally stained at MSU Veterinary Diagnostic Laboratory using the VENTANA HE600 system from Roche.

    Laser Capture Microdissection:

    Article Title: [2401.06169] Deep Learning model predicts the c-Kit-11 mutational status of canine cutaneous mast cell tumors by HE stained histological slides
    Article Snippet: .. PCR-based identification of c-Kit exon 11 Mutational status Neoplastic cells were isolated from FFPE MCTs with laser-capture microdissection (LCM) as previously described.24 The exon 11 of the c-Kit gene was amplified by PCR using the primer pair at the 5’ end of the exon 11 (PE1: 5’- CCATGTATGAAGTACAGTGGAAG-3’ sense, bp 1,657–1,680 of exon 11) and the 5’- end of intron 11 (PE2: 5’GTTCCCTAAAGTCATTGTTACACG-3’ anti-sense, nucleotides 43–66 of intron 11).3 A Mutation Detection PCR Kit (Qiagen) was used for the amplification.2 PCRs were prepared in a 25-μL total reaction volume, with 10 to 25 ng of LCM-extracted DNA and 5 pmol of each primer 0.5 U of Taq polymerase (Invitrogen, Carlsbad, CA), and final concentrations of 80 μM deoxynucleoside triphosphate, 2 mM MgCl2, 20 mM Tris–HCl, and 50 μl of KCl.24 Cycling conditions were as follows: 95 °C for 5 minutes; 35 cycles at 95 °C for 30 seconds, 58 °C for 90 seconds, 72 °C for 30 seconds; followed by 68 °C for 10 minutes.2 PCR products were visualized on the QIAxcel Capillary Electrophoresis System (Qiagen).2 .. To investigate the dependency of the mutational status prediction on the staining protocol, we applied two stains (Stain A, Stain B) to the same tissue section, which was de-stained in between both staining steps, using the following protocol: HE-stain A – Paraffin sections of all tumors were originally stained at MSU Veterinary Diagnostic Laboratory using the VENTANA HE600 system from Roche.

    Amplification:

    Article Title: [2401.06169] Deep Learning model predicts the c-Kit-11 mutational status of canine cutaneous mast cell tumors by HE stained histological slides
    Article Snippet: .. PCR-based identification of c-Kit exon 11 Mutational status Neoplastic cells were isolated from FFPE MCTs with laser-capture microdissection (LCM) as previously described.24 The exon 11 of the c-Kit gene was amplified by PCR using the primer pair at the 5’ end of the exon 11 (PE1: 5’- CCATGTATGAAGTACAGTGGAAG-3’ sense, bp 1,657–1,680 of exon 11) and the 5’- end of intron 11 (PE2: 5’GTTCCCTAAAGTCATTGTTACACG-3’ anti-sense, nucleotides 43–66 of intron 11).3 A Mutation Detection PCR Kit (Qiagen) was used for the amplification.2 PCRs were prepared in a 25-μL total reaction volume, with 10 to 25 ng of LCM-extracted DNA and 5 pmol of each primer 0.5 U of Taq polymerase (Invitrogen, Carlsbad, CA), and final concentrations of 80 μM deoxynucleoside triphosphate, 2 mM MgCl2, 20 mM Tris–HCl, and 50 μl of KCl.24 Cycling conditions were as follows: 95 °C for 5 minutes; 35 cycles at 95 °C for 30 seconds, 58 °C for 90 seconds, 72 °C for 30 seconds; followed by 68 °C for 10 minutes.2 PCR products were visualized on the QIAxcel Capillary Electrophoresis System (Qiagen).2 .. To investigate the dependency of the mutational status prediction on the staining protocol, we applied two stains (Stain A, Stain B) to the same tissue section, which was de-stained in between both staining steps, using the following protocol: HE-stain A – Paraffin sections of all tumors were originally stained at MSU Veterinary Diagnostic Laboratory using the VENTANA HE600 system from Roche.

    Article Title: Artificial intelligence can be trained to predict c-KIT -11 mutational status of canine mast cell tumors from hematoxylin and eosin-stained histological slides
    Article Snippet: .. A Mutation Detection PCR Kit (Qiagen) was used for the amplification. .. PCRs were prepared in a 25 μl total reaction volume, with 10 to 25 ng of extracted DNA; 5 pmol of each primer; 0.5 U of Taq polymerase (Invitrogen, Carlsbad, California); and final concentrations of 80 μm deoxynucleoside triphosphate, 2 mM MgCl 2 , 20 mM Tris-HCl, and 50 μl of KCl.

    Article Title: Internal Tandem Duplication of Exon 8 of c-kit Is Associated With Longer Total Survival in Canine Cutaneous Mast Cell Tumors.
    Article Snippet: Canine cutaneous mast cell tumors (ccMCTs) have a highly variable biological behavior and accurate prognostication is essential for therapeutic intervention.. Internal tandem duplications (ITD) of exon 11 are the most commonly detected c-kit mutation in ccMCTs and are associated with poor prognosis and increased cellular proliferation.. The prognostic value of detecting mutations in other exons of c-kit has not been systematically examined.

    Article Title: Artificial intelligence can be trained to predict c-KIT -11 mutational status of canine mast cell tumors from hematoxylin and eosin-stained histological slides.
    Article Snippet: .. PCR-Based Identification of ITD Mutation in the c-KIT Exon 11 The PCR-based identification of c-KIT-11-ITD was done at the Michigan State University as part of the MCT panel for routine diagnostics, as previously described.32 Exon 11 of the c-KIT gene was amplified by PCR using the primer pair at the 5’ end of the exon 11 (PE1: 5’-CCATGTATGAAGTACAGTGGAAG-3’ sense, bp 1657–1680 of exon 11) and the 5’ end of intron 11 (PE2: 5’GTTCCCTAAAGTCATTGTTACACG-3’ anti-sense, nucleotides 43–66 of intron 11).4 A Mutation Detection PCR Kit (Qiagen) was used for the amplification.3 PCRs were prepared in a 25 μl total reaction volume, with 10 to 25 ng of extracted DNA; 5 pmol of each primer; 0.5 U of Taq polymerase (Invitrogen, Carlsbad, California); and final concentrations of 80 μm deoxynucleoside triphosphate, 2 mM MgCl2, 20 mM Tris-HCl, and 50 μl of KCl.32 Cycling conditions were as follows: 95°C for 5 minutes; 35 cycles at 95°C for 30 seconds, 58°C for 90 seconds, 72°C for 30 seconds; followed by 68°C for 10 minutes.3 PCR products were visualized on the QIAxcel Capillary Electrophoresis System (Qiagen).3 Staining Variants To investigate the dependency of the mutational status prediction on the staining protocol, we applied 2 stains (stains A and B) to the same tissue slice, which was de-stained in-between the staining steps, using the following protocol: HE—stain A—Paraffin sections of all tumors were originally stained at Michigan State University Veterinary Diagnostic Laboratory using the Ventana HE600 system from Roche. .. Dry deparaffinization, rehydration, and HE staining were run according to a standard protocol11 and using Ventana HE 600 Hematoxylin (Roche, 07024282001) and Ventana HE 600 Eosin (Roche, 06544304001).

    Electrophoresis:

    Article Title: [2401.06169] Deep Learning model predicts the c-Kit-11 mutational status of canine cutaneous mast cell tumors by HE stained histological slides
    Article Snippet: .. PCR-based identification of c-Kit exon 11 Mutational status Neoplastic cells were isolated from FFPE MCTs with laser-capture microdissection (LCM) as previously described.24 The exon 11 of the c-Kit gene was amplified by PCR using the primer pair at the 5’ end of the exon 11 (PE1: 5’- CCATGTATGAAGTACAGTGGAAG-3’ sense, bp 1,657–1,680 of exon 11) and the 5’- end of intron 11 (PE2: 5’GTTCCCTAAAGTCATTGTTACACG-3’ anti-sense, nucleotides 43–66 of intron 11).3 A Mutation Detection PCR Kit (Qiagen) was used for the amplification.2 PCRs were prepared in a 25-μL total reaction volume, with 10 to 25 ng of LCM-extracted DNA and 5 pmol of each primer 0.5 U of Taq polymerase (Invitrogen, Carlsbad, CA), and final concentrations of 80 μM deoxynucleoside triphosphate, 2 mM MgCl2, 20 mM Tris–HCl, and 50 μl of KCl.24 Cycling conditions were as follows: 95 °C for 5 minutes; 35 cycles at 95 °C for 30 seconds, 58 °C for 90 seconds, 72 °C for 30 seconds; followed by 68 °C for 10 minutes.2 PCR products were visualized on the QIAxcel Capillary Electrophoresis System (Qiagen).2 .. To investigate the dependency of the mutational status prediction on the staining protocol, we applied two stains (Stain A, Stain B) to the same tissue section, which was de-stained in between both staining steps, using the following protocol: HE-stain A – Paraffin sections of all tumors were originally stained at MSU Veterinary Diagnostic Laboratory using the VENTANA HE600 system from Roche.

    Article Title: Artificial intelligence can be trained to predict c-KIT -11 mutational status of canine mast cell tumors from hematoxylin and eosin-stained histological slides.
    Article Snippet: .. PCR-Based Identification of ITD Mutation in the c-KIT Exon 11 The PCR-based identification of c-KIT-11-ITD was done at the Michigan State University as part of the MCT panel for routine diagnostics, as previously described.32 Exon 11 of the c-KIT gene was amplified by PCR using the primer pair at the 5’ end of the exon 11 (PE1: 5’-CCATGTATGAAGTACAGTGGAAG-3’ sense, bp 1657–1680 of exon 11) and the 5’ end of intron 11 (PE2: 5’GTTCCCTAAAGTCATTGTTACACG-3’ anti-sense, nucleotides 43–66 of intron 11).4 A Mutation Detection PCR Kit (Qiagen) was used for the amplification.3 PCRs were prepared in a 25 μl total reaction volume, with 10 to 25 ng of extracted DNA; 5 pmol of each primer; 0.5 U of Taq polymerase (Invitrogen, Carlsbad, California); and final concentrations of 80 μm deoxynucleoside triphosphate, 2 mM MgCl2, 20 mM Tris-HCl, and 50 μl of KCl.32 Cycling conditions were as follows: 95°C for 5 minutes; 35 cycles at 95°C for 30 seconds, 58°C for 90 seconds, 72°C for 30 seconds; followed by 68°C for 10 minutes.3 PCR products were visualized on the QIAxcel Capillary Electrophoresis System (Qiagen).3 Staining Variants To investigate the dependency of the mutational status prediction on the staining protocol, we applied 2 stains (stains A and B) to the same tissue slice, which was de-stained in-between the staining steps, using the following protocol: HE—stain A—Paraffin sections of all tumors were originally stained at Michigan State University Veterinary Diagnostic Laboratory using the Ventana HE600 system from Roche. .. Dry deparaffinization, rehydration, and HE staining were run according to a standard protocol11 and using Ventana HE 600 Hematoxylin (Roche, 07024282001) and Ventana HE 600 Eosin (Roche, 06544304001).

    Staining:

    Article Title: Artificial intelligence can be trained to predict c-KIT -11 mutational status of canine mast cell tumors from hematoxylin and eosin-stained histological slides.
    Article Snippet: .. PCR-Based Identification of ITD Mutation in the c-KIT Exon 11 The PCR-based identification of c-KIT-11-ITD was done at the Michigan State University as part of the MCT panel for routine diagnostics, as previously described.32 Exon 11 of the c-KIT gene was amplified by PCR using the primer pair at the 5’ end of the exon 11 (PE1: 5’-CCATGTATGAAGTACAGTGGAAG-3’ sense, bp 1657–1680 of exon 11) and the 5’ end of intron 11 (PE2: 5’GTTCCCTAAAGTCATTGTTACACG-3’ anti-sense, nucleotides 43–66 of intron 11).4 A Mutation Detection PCR Kit (Qiagen) was used for the amplification.3 PCRs were prepared in a 25 μl total reaction volume, with 10 to 25 ng of extracted DNA; 5 pmol of each primer; 0.5 U of Taq polymerase (Invitrogen, Carlsbad, California); and final concentrations of 80 μm deoxynucleoside triphosphate, 2 mM MgCl2, 20 mM Tris-HCl, and 50 μl of KCl.32 Cycling conditions were as follows: 95°C for 5 minutes; 35 cycles at 95°C for 30 seconds, 58°C for 90 seconds, 72°C for 30 seconds; followed by 68°C for 10 minutes.3 PCR products were visualized on the QIAxcel Capillary Electrophoresis System (Qiagen).3 Staining Variants To investigate the dependency of the mutational status prediction on the staining protocol, we applied 2 stains (stains A and B) to the same tissue slice, which was de-stained in-between the staining steps, using the following protocol: HE—stain A—Paraffin sections of all tumors were originally stained at Michigan State University Veterinary Diagnostic Laboratory using the Ventana HE600 system from Roche. .. Dry deparaffinization, rehydration, and HE staining were run according to a standard protocol11 and using Ventana HE 600 Hematoxylin (Roche, 07024282001) and Ventana HE 600 Eosin (Roche, 06544304001).

    Generated:

    Article Title: Development of a RACE-based RNA-Seq approach to characterize the T-cell receptor repertoire of porcine γδ T cells.
    Article Snippet: Recent data suggest that porcine γδ T cells exhibit a similar degree of functional plasticity as human and murine γδ T cells.. Due to the high frequency of TCR-γδ cells in blood and secondary lymphatic organs, the pig is an attractive model to study these cells, especially their combined features of the innate and the adaptive immune system.. Using a 5’ RACE-like approach, we translated a human/murine NGS library preparation strategy to capture full-length V-(D)-J TRG and TRD clonotypes in swine.



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    Generation and validation of Hsd17b3 knockout (KO) mice expressing HSD17B12 L234F , with <t>a</t> <t>mutation</t> in amino acid position 234 that prevents its ability to convert androstenedione to testosterone. (A) Sanger sequencing of mouse Hsd17b12 in wild-type and mutated animals. Point mutations in the DNA sequence were made to change the leucine (Leu) amino acid to a phenylalanine (Phe) amino acid, altering the amino acid sequence to replicate that seen in the human protein sequence. The mutated sequence is referred to as L234F. Created with BioRender.com . (B) Mutated Hsd17b12 mice ( Hsd17b12 L234F ) were cross-bred with Hsd17b3 KO mice to generate a mouse line incorporating both the Hsd17b3 null allele and the Hsd17b12 mutation. Created with BioRender.com . (C, D) Genotypes of mice were determined using standard <t>PCR.</t> (C) Presence of the Hsd17b3 +/+ (wild-type) allele was indicated by a 153-base pair (bp) band. The Hsd17b3 −/− ( Hsd17b3 KO) allele was indicated by a 7-bp deletion which showed a 146-bp band. (D) The presence or absence (+ or −) of the Hsd17b12 wild-type gene and the mutated Hsd17b12 (L234F) gene was identified by PCR products that were 436 and 432 bp, respectively. Interleukin-2 (IL-2) was used as a positive control in amplified samples, shown by a 324-bp band.
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    Generation and validation of Hsd17b3 knockout (KO) mice expressing HSD17B12 L234F , with <t>a</t> <t>mutation</t> in amino acid position 234 that prevents its ability to convert androstenedione to testosterone. (A) Sanger sequencing of mouse Hsd17b12 in wild-type and mutated animals. Point mutations in the DNA sequence were made to change the leucine (Leu) amino acid to a phenylalanine (Phe) amino acid, altering the amino acid sequence to replicate that seen in the human protein sequence. The mutated sequence is referred to as L234F. Created with BioRender.com . (B) Mutated Hsd17b12 mice ( Hsd17b12 L234F ) were cross-bred with Hsd17b3 KO mice to generate a mouse line incorporating both the Hsd17b3 null allele and the Hsd17b12 mutation. Created with BioRender.com . (C, D) Genotypes of mice were determined using standard <t>PCR.</t> (C) Presence of the Hsd17b3 +/+ (wild-type) allele was indicated by a 153-base pair (bp) band. The Hsd17b3 −/− ( Hsd17b3 KO) allele was indicated by a 7-bp deletion which showed a 146-bp band. (D) The presence or absence (+ or −) of the Hsd17b12 wild-type gene and the mutated Hsd17b12 (L234F) gene was identified by PCR products that were 436 and 432 bp, respectively. Interleukin-2 (IL-2) was used as a positive control in amplified samples, shown by a 324-bp band.
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    Generation and validation of Hsd17b3 knockout (KO) mice expressing HSD17B12 L234F , with a mutation in amino acid position 234 that prevents its ability to convert androstenedione to testosterone. (A) Sanger sequencing of mouse Hsd17b12 in wild-type and mutated animals. Point mutations in the DNA sequence were made to change the leucine (Leu) amino acid to a phenylalanine (Phe) amino acid, altering the amino acid sequence to replicate that seen in the human protein sequence. The mutated sequence is referred to as L234F. Created with BioRender.com . (B) Mutated Hsd17b12 mice ( Hsd17b12 L234F ) were cross-bred with Hsd17b3 KO mice to generate a mouse line incorporating both the Hsd17b3 null allele and the Hsd17b12 mutation. Created with BioRender.com . (C, D) Genotypes of mice were determined using standard PCR. (C) Presence of the Hsd17b3 +/+ (wild-type) allele was indicated by a 153-base pair (bp) band. The Hsd17b3 −/− ( Hsd17b3 KO) allele was indicated by a 7-bp deletion which showed a 146-bp band. (D) The presence or absence (+ or −) of the Hsd17b12 wild-type gene and the mutated Hsd17b12 (L234F) gene was identified by PCR products that were 436 and 432 bp, respectively. Interleukin-2 (IL-2) was used as a positive control in amplified samples, shown by a 324-bp band.

    Journal: Endocrinology

    Article Title: Functional Analysis of HSD17B3-Deficient Male Mice Reveals Roles for HSD17B7 and HSD17B12 in Testosterone Biosynthesis

    doi: 10.1210/endocr/bqaf078

    Figure Lengend Snippet: Generation and validation of Hsd17b3 knockout (KO) mice expressing HSD17B12 L234F , with a mutation in amino acid position 234 that prevents its ability to convert androstenedione to testosterone. (A) Sanger sequencing of mouse Hsd17b12 in wild-type and mutated animals. Point mutations in the DNA sequence were made to change the leucine (Leu) amino acid to a phenylalanine (Phe) amino acid, altering the amino acid sequence to replicate that seen in the human protein sequence. The mutated sequence is referred to as L234F. Created with BioRender.com . (B) Mutated Hsd17b12 mice ( Hsd17b12 L234F ) were cross-bred with Hsd17b3 KO mice to generate a mouse line incorporating both the Hsd17b3 null allele and the Hsd17b12 mutation. Created with BioRender.com . (C, D) Genotypes of mice were determined using standard PCR. (C) Presence of the Hsd17b3 +/+ (wild-type) allele was indicated by a 153-base pair (bp) band. The Hsd17b3 −/− ( Hsd17b3 KO) allele was indicated by a 7-bp deletion which showed a 146-bp band. (D) The presence or absence (+ or −) of the Hsd17b12 wild-type gene and the mutated Hsd17b12 (L234F) gene was identified by PCR products that were 436 and 432 bp, respectively. Interleukin-2 (IL-2) was used as a positive control in amplified samples, shown by a 324-bp band.

    Article Snippet: PCR was performed on gDNA extracts using a Type-it Mutation Detect PCR Kit (QIAGEN, VIC, Australia).

    Techniques: Biomarker Discovery, Knock-Out, Expressing, Mutagenesis, Sequencing, Positive Control, Amplification