The source of SYBR green master mix determines outcome of nucleic acid amplification reactions
Background Quantitative (q) PCR by amplification of nucleic acid with a fluorescent dye is widely used. Selection of adequate PCR reagents and devices is relevant to achieve reliable and consistent data. Our main objective was to test the robustness of different commercial SYBR green PCR mixes with...
Ausführliche Beschreibung
Autor*in: |
Yang, Jianxin [verfasserIn] |
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E-Artikel |
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Sprache: |
Englisch |
Erschienen: |
2016 |
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Anmerkung: |
© The Author(s) 2016 |
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Übergeordnetes Werk: |
Enthalten in: BMC Research Notes - London, 2008, 9(2016), 1 vom: 04. Juni |
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Übergeordnetes Werk: |
volume:9 ; year:2016 ; number:1 ; day:04 ; month:06 |
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DOI / URN: |
10.1186/s13104-016-2093-4 |
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Katalog-ID: |
SPR030316723 |
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520 | |a Background Quantitative (q) PCR by amplification of nucleic acid with a fluorescent dye is widely used. Selection of adequate PCR reagents and devices is relevant to achieve reliable and consistent data. Our main objective was to test the robustness of different commercial SYBR green PCR mixes with respect to specificity and sensitivity of the PCR assay, across various PCR machines (Light Cycler 96, ViiA7) and amplification protocols. Herein, we applied PCR protocols for determining mRNA transcript levels, DNA copy numbers, and DNA genotype. Results First, we set up 70 primer-based assays that targeted immune-related mRNA transcripts. Of the 70 assays 66 (94.3 %) resulted in a single melting curve peak, indicating specificity of the amplification, with PCR mixes from large vendors (Roche, ABI, Bio-Rad). But this was only seen when the PCR protocol that was indicated in the vendor’s guidelines for each particular mix was applied. When deviating from the prescribed protocol, suboptimal melting curves were most often seen when using Roche SYBR green. With respect to PCR yields, the use of ABI mix more often led to lower Cq values. Second, we set up 20 primer-selective PCR assays to target different insertion-deletion and single nucleotide polymorphism regions throughout the genome. The variation in delta Cq between positive and negative DNA samples among the PCR assays was the lowest when using ABI master mix. Finally, the quality of high resolution melting (HRM) assays for DNA genotyping was compared between four commercial HRM PCR mixes (Roche, Bioline, PCR Biosystems, ABI). Only Roche and ABI mixes produced optimal clusters of melting profiles that clearly distinguished genotype variants. Conclusions The current results show a preference for the use of ABI mix when it comes to obtaining higher sensitivity in cDNA analysis and a higher consistency among assays in distinguishing DNA genotypes among different individuals. For HRM assays, it is advisable to use master mix from a relatively large vendor. | ||
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10.1186/s13104-016-2093-4 doi (DE-627)SPR030316723 (SPR)s13104-016-2093-4-e DE-627 ger DE-627 rakwb eng Yang, Jianxin verfasserin aut The source of SYBR green master mix determines outcome of nucleic acid amplification reactions 2016 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2016 Background Quantitative (q) PCR by amplification of nucleic acid with a fluorescent dye is widely used. Selection of adequate PCR reagents and devices is relevant to achieve reliable and consistent data. Our main objective was to test the robustness of different commercial SYBR green PCR mixes with respect to specificity and sensitivity of the PCR assay, across various PCR machines (Light Cycler 96, ViiA7) and amplification protocols. Herein, we applied PCR protocols for determining mRNA transcript levels, DNA copy numbers, and DNA genotype. Results First, we set up 70 primer-based assays that targeted immune-related mRNA transcripts. Of the 70 assays 66 (94.3 %) resulted in a single melting curve peak, indicating specificity of the amplification, with PCR mixes from large vendors (Roche, ABI, Bio-Rad). But this was only seen when the PCR protocol that was indicated in the vendor’s guidelines for each particular mix was applied. When deviating from the prescribed protocol, suboptimal melting curves were most often seen when using Roche SYBR green. With respect to PCR yields, the use of ABI mix more often led to lower Cq values. Second, we set up 20 primer-selective PCR assays to target different insertion-deletion and single nucleotide polymorphism regions throughout the genome. The variation in delta Cq between positive and negative DNA samples among the PCR assays was the lowest when using ABI master mix. Finally, the quality of high resolution melting (HRM) assays for DNA genotyping was compared between four commercial HRM PCR mixes (Roche, Bioline, PCR Biosystems, ABI). Only Roche and ABI mixes produced optimal clusters of melting profiles that clearly distinguished genotype variants. Conclusions The current results show a preference for the use of ABI mix when it comes to obtaining higher sensitivity in cDNA analysis and a higher consistency among assays in distinguishing DNA genotypes among different individuals. For HRM assays, it is advisable to use master mix from a relatively large vendor. Quantitative PCR (dpeaa)DE-He213 Specificity (dpeaa)DE-He213 Genomic DNA (dpeaa)DE-He213 mRNA (dpeaa)DE-He213 High resolution melting (dpeaa)DE-He213 Performance (dpeaa)DE-He213 Kemps-Mols, Berit aut Spruyt-Gerritse, Marijke aut Anholts, Jacqueline aut Claas, Frans aut Eikmans, Michael aut Enthalten in BMC Research Notes London, 2008 9(2016), 1 vom: 04. Juni (DE-627)559431805 (DE-600)2413336-X 1756-0500 nnns volume:9 year:2016 number:1 day:04 month:06 https://dx.doi.org/10.1186/s13104-016-2093-4 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 9 2016 1 04 06 |
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10.1186/s13104-016-2093-4 doi (DE-627)SPR030316723 (SPR)s13104-016-2093-4-e DE-627 ger DE-627 rakwb eng Yang, Jianxin verfasserin aut The source of SYBR green master mix determines outcome of nucleic acid amplification reactions 2016 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2016 Background Quantitative (q) PCR by amplification of nucleic acid with a fluorescent dye is widely used. Selection of adequate PCR reagents and devices is relevant to achieve reliable and consistent data. Our main objective was to test the robustness of different commercial SYBR green PCR mixes with respect to specificity and sensitivity of the PCR assay, across various PCR machines (Light Cycler 96, ViiA7) and amplification protocols. Herein, we applied PCR protocols for determining mRNA transcript levels, DNA copy numbers, and DNA genotype. Results First, we set up 70 primer-based assays that targeted immune-related mRNA transcripts. Of the 70 assays 66 (94.3 %) resulted in a single melting curve peak, indicating specificity of the amplification, with PCR mixes from large vendors (Roche, ABI, Bio-Rad). But this was only seen when the PCR protocol that was indicated in the vendor’s guidelines for each particular mix was applied. When deviating from the prescribed protocol, suboptimal melting curves were most often seen when using Roche SYBR green. With respect to PCR yields, the use of ABI mix more often led to lower Cq values. Second, we set up 20 primer-selective PCR assays to target different insertion-deletion and single nucleotide polymorphism regions throughout the genome. The variation in delta Cq between positive and negative DNA samples among the PCR assays was the lowest when using ABI master mix. Finally, the quality of high resolution melting (HRM) assays for DNA genotyping was compared between four commercial HRM PCR mixes (Roche, Bioline, PCR Biosystems, ABI). Only Roche and ABI mixes produced optimal clusters of melting profiles that clearly distinguished genotype variants. Conclusions The current results show a preference for the use of ABI mix when it comes to obtaining higher sensitivity in cDNA analysis and a higher consistency among assays in distinguishing DNA genotypes among different individuals. For HRM assays, it is advisable to use master mix from a relatively large vendor. Quantitative PCR (dpeaa)DE-He213 Specificity (dpeaa)DE-He213 Genomic DNA (dpeaa)DE-He213 mRNA (dpeaa)DE-He213 High resolution melting (dpeaa)DE-He213 Performance (dpeaa)DE-He213 Kemps-Mols, Berit aut Spruyt-Gerritse, Marijke aut Anholts, Jacqueline aut Claas, Frans aut Eikmans, Michael aut Enthalten in BMC Research Notes London, 2008 9(2016), 1 vom: 04. Juni (DE-627)559431805 (DE-600)2413336-X 1756-0500 nnns volume:9 year:2016 number:1 day:04 month:06 https://dx.doi.org/10.1186/s13104-016-2093-4 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 9 2016 1 04 06 |
allfields_unstemmed |
10.1186/s13104-016-2093-4 doi (DE-627)SPR030316723 (SPR)s13104-016-2093-4-e DE-627 ger DE-627 rakwb eng Yang, Jianxin verfasserin aut The source of SYBR green master mix determines outcome of nucleic acid amplification reactions 2016 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2016 Background Quantitative (q) PCR by amplification of nucleic acid with a fluorescent dye is widely used. Selection of adequate PCR reagents and devices is relevant to achieve reliable and consistent data. Our main objective was to test the robustness of different commercial SYBR green PCR mixes with respect to specificity and sensitivity of the PCR assay, across various PCR machines (Light Cycler 96, ViiA7) and amplification protocols. Herein, we applied PCR protocols for determining mRNA transcript levels, DNA copy numbers, and DNA genotype. Results First, we set up 70 primer-based assays that targeted immune-related mRNA transcripts. Of the 70 assays 66 (94.3 %) resulted in a single melting curve peak, indicating specificity of the amplification, with PCR mixes from large vendors (Roche, ABI, Bio-Rad). But this was only seen when the PCR protocol that was indicated in the vendor’s guidelines for each particular mix was applied. When deviating from the prescribed protocol, suboptimal melting curves were most often seen when using Roche SYBR green. With respect to PCR yields, the use of ABI mix more often led to lower Cq values. Second, we set up 20 primer-selective PCR assays to target different insertion-deletion and single nucleotide polymorphism regions throughout the genome. The variation in delta Cq between positive and negative DNA samples among the PCR assays was the lowest when using ABI master mix. Finally, the quality of high resolution melting (HRM) assays for DNA genotyping was compared between four commercial HRM PCR mixes (Roche, Bioline, PCR Biosystems, ABI). Only Roche and ABI mixes produced optimal clusters of melting profiles that clearly distinguished genotype variants. Conclusions The current results show a preference for the use of ABI mix when it comes to obtaining higher sensitivity in cDNA analysis and a higher consistency among assays in distinguishing DNA genotypes among different individuals. For HRM assays, it is advisable to use master mix from a relatively large vendor. Quantitative PCR (dpeaa)DE-He213 Specificity (dpeaa)DE-He213 Genomic DNA (dpeaa)DE-He213 mRNA (dpeaa)DE-He213 High resolution melting (dpeaa)DE-He213 Performance (dpeaa)DE-He213 Kemps-Mols, Berit aut Spruyt-Gerritse, Marijke aut Anholts, Jacqueline aut Claas, Frans aut Eikmans, Michael aut Enthalten in BMC Research Notes London, 2008 9(2016), 1 vom: 04. Juni (DE-627)559431805 (DE-600)2413336-X 1756-0500 nnns volume:9 year:2016 number:1 day:04 month:06 https://dx.doi.org/10.1186/s13104-016-2093-4 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 9 2016 1 04 06 |
allfieldsGer |
10.1186/s13104-016-2093-4 doi (DE-627)SPR030316723 (SPR)s13104-016-2093-4-e DE-627 ger DE-627 rakwb eng Yang, Jianxin verfasserin aut The source of SYBR green master mix determines outcome of nucleic acid amplification reactions 2016 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2016 Background Quantitative (q) PCR by amplification of nucleic acid with a fluorescent dye is widely used. Selection of adequate PCR reagents and devices is relevant to achieve reliable and consistent data. Our main objective was to test the robustness of different commercial SYBR green PCR mixes with respect to specificity and sensitivity of the PCR assay, across various PCR machines (Light Cycler 96, ViiA7) and amplification protocols. Herein, we applied PCR protocols for determining mRNA transcript levels, DNA copy numbers, and DNA genotype. Results First, we set up 70 primer-based assays that targeted immune-related mRNA transcripts. Of the 70 assays 66 (94.3 %) resulted in a single melting curve peak, indicating specificity of the amplification, with PCR mixes from large vendors (Roche, ABI, Bio-Rad). But this was only seen when the PCR protocol that was indicated in the vendor’s guidelines for each particular mix was applied. When deviating from the prescribed protocol, suboptimal melting curves were most often seen when using Roche SYBR green. With respect to PCR yields, the use of ABI mix more often led to lower Cq values. Second, we set up 20 primer-selective PCR assays to target different insertion-deletion and single nucleotide polymorphism regions throughout the genome. The variation in delta Cq between positive and negative DNA samples among the PCR assays was the lowest when using ABI master mix. Finally, the quality of high resolution melting (HRM) assays for DNA genotyping was compared between four commercial HRM PCR mixes (Roche, Bioline, PCR Biosystems, ABI). Only Roche and ABI mixes produced optimal clusters of melting profiles that clearly distinguished genotype variants. Conclusions The current results show a preference for the use of ABI mix when it comes to obtaining higher sensitivity in cDNA analysis and a higher consistency among assays in distinguishing DNA genotypes among different individuals. For HRM assays, it is advisable to use master mix from a relatively large vendor. Quantitative PCR (dpeaa)DE-He213 Specificity (dpeaa)DE-He213 Genomic DNA (dpeaa)DE-He213 mRNA (dpeaa)DE-He213 High resolution melting (dpeaa)DE-He213 Performance (dpeaa)DE-He213 Kemps-Mols, Berit aut Spruyt-Gerritse, Marijke aut Anholts, Jacqueline aut Claas, Frans aut Eikmans, Michael aut Enthalten in BMC Research Notes London, 2008 9(2016), 1 vom: 04. Juni (DE-627)559431805 (DE-600)2413336-X 1756-0500 nnns volume:9 year:2016 number:1 day:04 month:06 https://dx.doi.org/10.1186/s13104-016-2093-4 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 9 2016 1 04 06 |
allfieldsSound |
10.1186/s13104-016-2093-4 doi (DE-627)SPR030316723 (SPR)s13104-016-2093-4-e DE-627 ger DE-627 rakwb eng Yang, Jianxin verfasserin aut The source of SYBR green master mix determines outcome of nucleic acid amplification reactions 2016 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2016 Background Quantitative (q) PCR by amplification of nucleic acid with a fluorescent dye is widely used. Selection of adequate PCR reagents and devices is relevant to achieve reliable and consistent data. Our main objective was to test the robustness of different commercial SYBR green PCR mixes with respect to specificity and sensitivity of the PCR assay, across various PCR machines (Light Cycler 96, ViiA7) and amplification protocols. Herein, we applied PCR protocols for determining mRNA transcript levels, DNA copy numbers, and DNA genotype. Results First, we set up 70 primer-based assays that targeted immune-related mRNA transcripts. Of the 70 assays 66 (94.3 %) resulted in a single melting curve peak, indicating specificity of the amplification, with PCR mixes from large vendors (Roche, ABI, Bio-Rad). But this was only seen when the PCR protocol that was indicated in the vendor’s guidelines for each particular mix was applied. When deviating from the prescribed protocol, suboptimal melting curves were most often seen when using Roche SYBR green. With respect to PCR yields, the use of ABI mix more often led to lower Cq values. Second, we set up 20 primer-selective PCR assays to target different insertion-deletion and single nucleotide polymorphism regions throughout the genome. The variation in delta Cq between positive and negative DNA samples among the PCR assays was the lowest when using ABI master mix. Finally, the quality of high resolution melting (HRM) assays for DNA genotyping was compared between four commercial HRM PCR mixes (Roche, Bioline, PCR Biosystems, ABI). Only Roche and ABI mixes produced optimal clusters of melting profiles that clearly distinguished genotype variants. Conclusions The current results show a preference for the use of ABI mix when it comes to obtaining higher sensitivity in cDNA analysis and a higher consistency among assays in distinguishing DNA genotypes among different individuals. For HRM assays, it is advisable to use master mix from a relatively large vendor. Quantitative PCR (dpeaa)DE-He213 Specificity (dpeaa)DE-He213 Genomic DNA (dpeaa)DE-He213 mRNA (dpeaa)DE-He213 High resolution melting (dpeaa)DE-He213 Performance (dpeaa)DE-He213 Kemps-Mols, Berit aut Spruyt-Gerritse, Marijke aut Anholts, Jacqueline aut Claas, Frans aut Eikmans, Michael aut Enthalten in BMC Research Notes London, 2008 9(2016), 1 vom: 04. Juni (DE-627)559431805 (DE-600)2413336-X 1756-0500 nnns volume:9 year:2016 number:1 day:04 month:06 https://dx.doi.org/10.1186/s13104-016-2093-4 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 9 2016 1 04 06 |
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Yang, Jianxin @@aut@@ Kemps-Mols, Berit @@aut@@ Spruyt-Gerritse, Marijke @@aut@@ Anholts, Jacqueline @@aut@@ Claas, Frans @@aut@@ Eikmans, Michael @@aut@@ |
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Yang, Jianxin |
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The source of SYBR green master mix determines outcome of nucleic acid amplification reactions Quantitative PCR (dpeaa)DE-He213 Specificity (dpeaa)DE-He213 Genomic DNA (dpeaa)DE-He213 mRNA (dpeaa)DE-He213 High resolution melting (dpeaa)DE-He213 Performance (dpeaa)DE-He213 |
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source of sybr green master mix determines outcome of nucleic acid amplification reactions |
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The source of SYBR green master mix determines outcome of nucleic acid amplification reactions |
abstract |
Background Quantitative (q) PCR by amplification of nucleic acid with a fluorescent dye is widely used. Selection of adequate PCR reagents and devices is relevant to achieve reliable and consistent data. Our main objective was to test the robustness of different commercial SYBR green PCR mixes with respect to specificity and sensitivity of the PCR assay, across various PCR machines (Light Cycler 96, ViiA7) and amplification protocols. Herein, we applied PCR protocols for determining mRNA transcript levels, DNA copy numbers, and DNA genotype. Results First, we set up 70 primer-based assays that targeted immune-related mRNA transcripts. Of the 70 assays 66 (94.3 %) resulted in a single melting curve peak, indicating specificity of the amplification, with PCR mixes from large vendors (Roche, ABI, Bio-Rad). But this was only seen when the PCR protocol that was indicated in the vendor’s guidelines for each particular mix was applied. When deviating from the prescribed protocol, suboptimal melting curves were most often seen when using Roche SYBR green. With respect to PCR yields, the use of ABI mix more often led to lower Cq values. Second, we set up 20 primer-selective PCR assays to target different insertion-deletion and single nucleotide polymorphism regions throughout the genome. The variation in delta Cq between positive and negative DNA samples among the PCR assays was the lowest when using ABI master mix. Finally, the quality of high resolution melting (HRM) assays for DNA genotyping was compared between four commercial HRM PCR mixes (Roche, Bioline, PCR Biosystems, ABI). Only Roche and ABI mixes produced optimal clusters of melting profiles that clearly distinguished genotype variants. Conclusions The current results show a preference for the use of ABI mix when it comes to obtaining higher sensitivity in cDNA analysis and a higher consistency among assays in distinguishing DNA genotypes among different individuals. For HRM assays, it is advisable to use master mix from a relatively large vendor. © The Author(s) 2016 |
abstractGer |
Background Quantitative (q) PCR by amplification of nucleic acid with a fluorescent dye is widely used. Selection of adequate PCR reagents and devices is relevant to achieve reliable and consistent data. Our main objective was to test the robustness of different commercial SYBR green PCR mixes with respect to specificity and sensitivity of the PCR assay, across various PCR machines (Light Cycler 96, ViiA7) and amplification protocols. Herein, we applied PCR protocols for determining mRNA transcript levels, DNA copy numbers, and DNA genotype. Results First, we set up 70 primer-based assays that targeted immune-related mRNA transcripts. Of the 70 assays 66 (94.3 %) resulted in a single melting curve peak, indicating specificity of the amplification, with PCR mixes from large vendors (Roche, ABI, Bio-Rad). But this was only seen when the PCR protocol that was indicated in the vendor’s guidelines for each particular mix was applied. When deviating from the prescribed protocol, suboptimal melting curves were most often seen when using Roche SYBR green. With respect to PCR yields, the use of ABI mix more often led to lower Cq values. Second, we set up 20 primer-selective PCR assays to target different insertion-deletion and single nucleotide polymorphism regions throughout the genome. The variation in delta Cq between positive and negative DNA samples among the PCR assays was the lowest when using ABI master mix. Finally, the quality of high resolution melting (HRM) assays for DNA genotyping was compared between four commercial HRM PCR mixes (Roche, Bioline, PCR Biosystems, ABI). Only Roche and ABI mixes produced optimal clusters of melting profiles that clearly distinguished genotype variants. Conclusions The current results show a preference for the use of ABI mix when it comes to obtaining higher sensitivity in cDNA analysis and a higher consistency among assays in distinguishing DNA genotypes among different individuals. For HRM assays, it is advisable to use master mix from a relatively large vendor. © The Author(s) 2016 |
abstract_unstemmed |
Background Quantitative (q) PCR by amplification of nucleic acid with a fluorescent dye is widely used. Selection of adequate PCR reagents and devices is relevant to achieve reliable and consistent data. Our main objective was to test the robustness of different commercial SYBR green PCR mixes with respect to specificity and sensitivity of the PCR assay, across various PCR machines (Light Cycler 96, ViiA7) and amplification protocols. Herein, we applied PCR protocols for determining mRNA transcript levels, DNA copy numbers, and DNA genotype. Results First, we set up 70 primer-based assays that targeted immune-related mRNA transcripts. Of the 70 assays 66 (94.3 %) resulted in a single melting curve peak, indicating specificity of the amplification, with PCR mixes from large vendors (Roche, ABI, Bio-Rad). But this was only seen when the PCR protocol that was indicated in the vendor’s guidelines for each particular mix was applied. When deviating from the prescribed protocol, suboptimal melting curves were most often seen when using Roche SYBR green. With respect to PCR yields, the use of ABI mix more often led to lower Cq values. Second, we set up 20 primer-selective PCR assays to target different insertion-deletion and single nucleotide polymorphism regions throughout the genome. The variation in delta Cq between positive and negative DNA samples among the PCR assays was the lowest when using ABI master mix. Finally, the quality of high resolution melting (HRM) assays for DNA genotyping was compared between four commercial HRM PCR mixes (Roche, Bioline, PCR Biosystems, ABI). Only Roche and ABI mixes produced optimal clusters of melting profiles that clearly distinguished genotype variants. Conclusions The current results show a preference for the use of ABI mix when it comes to obtaining higher sensitivity in cDNA analysis and a higher consistency among assays in distinguishing DNA genotypes among different individuals. For HRM assays, it is advisable to use master mix from a relatively large vendor. © The Author(s) 2016 |
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The source of SYBR green master mix determines outcome of nucleic acid amplification reactions |
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Selection of adequate PCR reagents and devices is relevant to achieve reliable and consistent data. Our main objective was to test the robustness of different commercial SYBR green PCR mixes with respect to specificity and sensitivity of the PCR assay, across various PCR machines (Light Cycler 96, ViiA7) and amplification protocols. Herein, we applied PCR protocols for determining mRNA transcript levels, DNA copy numbers, and DNA genotype. Results First, we set up 70 primer-based assays that targeted immune-related mRNA transcripts. Of the 70 assays 66 (94.3 %) resulted in a single melting curve peak, indicating specificity of the amplification, with PCR mixes from large vendors (Roche, ABI, Bio-Rad). But this was only seen when the PCR protocol that was indicated in the vendor’s guidelines for each particular mix was applied. When deviating from the prescribed protocol, suboptimal melting curves were most often seen when using Roche SYBR green. With respect to PCR yields, the use of ABI mix more often led to lower Cq values. Second, we set up 20 primer-selective PCR assays to target different insertion-deletion and single nucleotide polymorphism regions throughout the genome. The variation in delta Cq between positive and negative DNA samples among the PCR assays was the lowest when using ABI master mix. Finally, the quality of high resolution melting (HRM) assays for DNA genotyping was compared between four commercial HRM PCR mixes (Roche, Bioline, PCR Biosystems, ABI). Only Roche and ABI mixes produced optimal clusters of melting profiles that clearly distinguished genotype variants. Conclusions The current results show a preference for the use of ABI mix when it comes to obtaining higher sensitivity in cDNA analysis and a higher consistency among assays in distinguishing DNA genotypes among different individuals. 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