high efficiency qpcr with primetime® gene expression master mix from idt
TRANSCRIPT
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Dr Erik Wendlandt Scientific Applications Specialist9/28/2016
High efficiency qPCR with PrimeTime® Gene Expression Master Mix
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Outline• Review of 5′-nuclease assays• PrimeTime® Gene Expression Master Mix components• Performance of the PrimeTime Gene Expression Master Mix
• With IDT assays• With commercially available assays
• PrimeTime Gene Expression Master Mix stability• Singleplex vs. multiplexing applications
• Considerations for multiplexing• Useful tools for multiplexing
• Oligo Analyzer®, Multiplex Dye selection Tool• Standard vs. fast cycling conditions
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Primers onlyFor use with intercalating dyes, such as SYBR® Green
Primers and probeFor use in the 5′-nuclease assay
5′-nuclease assays vs. Intercalating-dye
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Primers onlyFor use with intercalating dyes, such as SYBR® Green
Primers and probeFor use in the 5′-nuclease assay
5′-nuclease assays vs. Intercalating-dye
The PrimeTime® Gene Expression Master Mix has been optimized for use with 5 -nuclease assays′
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Outline• Review of 5′-nuclease assays• PrimeTime® Gene Expression Master Mix components• Performance of the PrimeTime Gene Expression Master Mix
• With IDT assays• With commercially available assays
• PrimeTime Gene Expression Master Mix stability• Singleplex vs. multiplexing applications
• Considerations for multiplexing• Useful tools for multiplexing
• Oligo Analyzer ®, Multiplex Dye selection Tool• Standard vs. fast cycling conditions
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PrimeTime® Gene Expression Master Mix
Ready-to-use, 2X concentrated master mix, designed for use in 2-step, probe-based RT-qPCR assays.
• Antibody-mediated, hot-start DNA polymerase • dNTPs • MgCl2 • Enhancers and stabilizers
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• PrimeTime Gene Expression Master Mix has been formulated to give optimal performance with PrimeTime qPCR Assays
• Predesigned PrimeTime qPCR Assays are available for human, mouse, and rat • Custom qPCR design tools are also
available• Use the same master mix for
singleplex/multiplex, fast/standard cycling conditions
• Exceptional benchtop stability • Obtain consistent results from each batch
for high-throughput and overnight runs
Why use the PrimeTime® Gene Expression Master Mix?
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www.idtdna.com/order/Predesignedassay.aspx
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Outline• Review of 5′-nuclease assays• PrimeTime® Gene Expression Master Mix components• Performance of the PrimeTime Gene Expression Master Mix
• With IDT assays• With commercially available assays
• PrimeTime Gene Expression Master Mix stability• Singleplex vs. multiplexing applications
• Considerations for multiplexing• Useful tools for multiplexing
• Oligo Analyzer®, Multiplex Dye selection Tool• Standard vs. fast cycling conditions
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The PrimeTime® Gene Expression Master Mix outperforms other commercially available master mixes
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Comparison of IDT PrimeTime® Gene Expression Master Mix and commercially available master mixqPCR assays consisting of:• PrimeTime Gene Expression Master Mix
with reference dye, or commercially available master mix
• Varying amounts of cDNA (0.016–50 ng) or gBlocks® Gene Fragments (107–101 copies)
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The PrimeTime® Gene Expression Master Mix outperforms other commercially available master mixes
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Comparison of IDT PrimeTime® Gene Expression Master Mix and commercially available master mixqPCR assays consisting of:• PrimeTime ® Gene Expression Master Mix
with reference dye, or commercially available master mix
• Varying amounts of cDNA (0.016–50 ng) or gBlocks® Gene Fragments (107–101 copies)
The PrimeTime® Gene Expression Master Mix consistently outperforms other commercially available master mixes
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Successful singleplex and duplex amplification using PrimeTime® Gene Expression Master Mix
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Singleplex and duplex PCR reactions were performed with: • PrimeTime Gene Expression Master Mix
with reference dye• Assays:
– Predesigned PrimeTime qPCR HPRT AssayAssay ID: Hs.PT.58v.45621572
– Predesigned PrimeTime qPCR GUSB Assay Assay ID: Hs.PT.58v.27737538
• gBlocks® Gene Fragments 107–101 copies
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Successful singleplex and duplex amplification using PrimeTime® Gene Expression Master Mix
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Singleplex and duplex PCR reactions were performed with: • PrimeTime Gene Expression Master Mix
with reference dye• Assays:
– Predesigned PrimeTime qPCR HPRT AssayAssay ID: Hs.PT.58v.45621572
– Predesigned PrimeTime qPCR GUSB Assay Assay ID: Hs.PT.58v.27737538
• gBlocks® Gene Fragments 107–101 copies
Efficient amplification when used with PrimeTime® qPCR assays
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PrimeTime® Gene Expression Master Mix also works well with assays purchased from other vendors
Singleplex and duplex PCR reactions were performed with: • PrimeTime Gene Expression Master
Mix with reference dye• Assays:
– Inventoried HPRT and GUSB qPCR assays from supplier AcDNA Template 0.0032–50 ng
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PrimeTime® Gene Expression Master Mix also works well with assays purchased from other vendors
Singleplex and duplex PCR reactions were performed with: • PrimeTime Gene Expression Master
Mix with reference dye• Assays:
– Inventoried HPRT and GUSB qPCR assays from supplier AcDNA Template 0.0032–50 ng
Efficient amplification when used with commercially available qPCR assays
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Outline• Review of 5′-nuclease assays• PrimeTime® Gene Expression Master Mix components• Performance of the PrimeTime Gene Expression Master Mix
• With IDT assays• With commercially available assays
• PrimeTime Gene Expression Master Mix stability• Singleplex vs. multiplexing applications
• Considerations for multiplexing• Useful tools for multiplexing
• Oligo Analyzer®, Multiplex Dye selection Tool• Standard vs. fast cycling conditions
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Reliable results using different batches of master mix: PCR held at room temperature for 0, 24, 48, and 72 hr before cycling qPCR assays consisting of:• PrimeTime® Gene Expression Master Mix,
reference dye• Varying amounts of gBlocks® Gene
Fragments107–102 copies
Consistent results for high-throughput qPCR
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Consistent results for high throughput qPCR
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Reliable results using different batches of master mix: PCR held at room temperature for 0, 24, 48, and 72 hr before cyclingqPCR assays consisting of:• PrimeTime® Gene Expression Master Mix,
reference dye,• Varying amounts of gBlocks® Gene
Fragments 107–102 copies
The PrimeTime® Gene Expression Master Mix exhibits consistent and precise results at room temperature for up to 72 hr
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Consistent results under thermal stress
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Reliable results using different batches of master mix: 0, 1, 3, 5, and 7 days at 50ºC. qPCR assays consisting of:• PrimeTime® Gene Expression Master Mix,
reference dye• Varying amounts of gBlocks® Gene
Fragments 107–102 copies
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Consistent results under thermal stress
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Consistent results under thermal stress
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The PrimeTime® Gene Expression Master Mix exhibits consistent performance after extended heat-stress
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PrimeTime® Gene Expression Master Mix is stable after international shipment under ambient conditions
21Data courtesy of Ticiane H Santa Rita and Dr Gustavo B Barra, Laboratório Sabin de Análises Clínicas, Brasília, DF
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PrimeTime® Gene Expression Master Mix is stable after international shipment under ambient conditions
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Shipping conditions do not affect performance of the PrimeTime® Gene Expression Master Mix
Data courtesy of Ticiane H Santa Rita and Dr Gustavo B Barra, Laboratório Sabin de Análises Clínicas, Brasília, DF
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Consistent results upon multiple freeze-thaw cycle
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Reliable results using different batches of master mix after 0, 5, 10, and 20 freeze-thaw cyclesqPCR assays consisting of:• PrimeTime® Gene Expression Master Mix,
reference dye• Varying amounts of gBlocks® Gene
Fragments 107–102 copies
![Page 24: High efficiency qPCR with PrimeTime® Gene Expression Master Mix from IDT](https://reader034.vdocuments.us/reader034/viewer/2022051708/58a636ef1a28ab68118b5251/html5/thumbnails/24.jpg)
Consistent results upon multiple freeze-thaw cycle
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Reliable results using different batches of master mix after 0, 5, 10, and 20 freeze-thaw cyclesqPCR assays consisting of:• PrimeTime® Gene Expression Master Mix,
reference dye,• Varying amounts of gBlocks® Gene
Fragments 107–102 copies
Multiple freeze-thaw cycles do not affect the functionality of the master mix
![Page 25: High efficiency qPCR with PrimeTime® Gene Expression Master Mix from IDT](https://reader034.vdocuments.us/reader034/viewer/2022051708/58a636ef1a28ab68118b5251/html5/thumbnails/25.jpg)
Outline• Review of 5′-nuclease assays• PrimeTime® Gene Expression Master Mix components• Performance of the PrimeTime Gene Expression Master Mix
• With IDT assays• With commercially available assays
• PrimeTime Gene Expression Master Mix stability• Singleplex vs. multiplexing applications
• Considerations for multiplexing• Useful tools for multiplexing
• Oligo Analyzer®, Multiplex Dye selection Tool• Standard vs. fast cycling conditions
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Considerations for multiplexing
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• Advantages• Save reagent/master mix costs• Analyze a large number of targets in a single reaction• Maximize usage of rare samples
• Considerations• Not all master mixes are formulated with enough polymerase and dNTPs to support
multiplexing—the PrimeTime® Gene Expression Master Mix is compatible with multiplexing• Need high efficiency assay designs for multiplexing
• Validate and optimize individual assays prior to multiplexing• Primers and probes must be assessed for interactions• Carefully select dyes for each assay
• Match the lowest copy target with the brightest fluorophore (FAM)• Lowered plateau in a multiplex vs. singleplex is normal
• Cq values should remain consistent between singleplex vs. multiplex
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Multiplexing with the PrimeTime® Gene Expression Master Mix(4-Plex)
Multiplex setupqPCR assays consisting of:• PrimeTime® Gene
Expression Master Mix, reference dye
• Varying amounts of cDNA 0.016–50 ng
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Multiplexing with the PrimeTime® Gene Expression Master Mix(Overlay)
FAM
Singleplex
Yakima Yellow Cy5 Cy5.5
Singleplex
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Multiplexing with the PrimeTime® Gene Expression Master Mix(Overlay)
FAM
Singleplex
Yakima Yellow Cy5 Cy5.5
Singleplex
Efficient amplification under singleplex and multiplex conditions
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Outline• Review of 5′-nuclease assays• PrimeTime® Gene Expression Master Mix components• Performance of the PrimeTime Gene Expression Master Mix
• With IDT assays• With commercially available assays
• PrimeTime Gene Expression Master Mix stability• Singleplex vs. multiplexing applications
• Considerations for multiplexing• Useful tools for multiplexing
• Oligo Analyzer®, Multiplex Dye selection Tool• Standard vs. fast cycling conditions
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Primer-dimer analysis with the Oligo Analyzer® Tool
c
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c
Primer-dimer analysis with the Oligo Analyzer® Tool
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Primer-dimer analysis with the Oligo Analyzer® Tool
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Primer-dimer analysis with the Oligo Analyzer® Tool
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Primer-dimer analysis with the Oligo Analyzer® Tool
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Primer-dimer analysis with the Oligo Analyzer® Tool
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Primer-dimer analysis with the Oligo Analyzer® Tool
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Primer-dimer analysis with the Oligo Analyzer® Tool
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PrimeTime® Multiplex Dye Selection Tool
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PrimeTime® Multiplex Dye Selection Tool
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PrimeTime® Multiplex Dye Selection Tool
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PrimeTime® Multiplex Dye Selection Tool
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PrimeTime® Multiplex Dye Selection Tool
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PrimeTime® Multiplex Dye Selection Tool
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PrimeTime® Multiplex Dye Selection Tool
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Outline• Review of 5′-nuclease assays• PrimeTime® Gene Expression Master Mix components• Performance of the PrimeTime Gene Expression Master Mix
• With IDT assays• With commercially available assays
• PrimeTime Gene Expression Master Mix stability• Singleplex vs. multiplexing applications
• Considerations for multiplexing• Useful tools for multiplexing
• Oligo Analyzer®, Multiplex Dye selection Tool• Standard vs. fast cycling conditions
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Efficiency of qPCR assays under fast and standard cycling conditions using PrimeTime® Gene Expression Master Mix
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Standard or fast cycling conditionsqPCR assays consisting of:• PrimeTime Gene Expression
Master Mix, reference dye• Varying amounts of cDNA 0.016–
50 ng
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Efficiency of qPCR assays under fast and standard cycling conditions using PrimeTime® Gene Expression Master Mix
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Standard or fast cycling conditionsqPCR assays consisting of:• PrimeTime Gene Expression
Master Mix, reference dye,• Varying amounts of cDNA 0.016–
50 ng
![Page 49: High efficiency qPCR with PrimeTime® Gene Expression Master Mix from IDT](https://reader034.vdocuments.us/reader034/viewer/2022051708/58a636ef1a28ab68118b5251/html5/thumbnails/49.jpg)
Efficiency of qPCR assays under fast and standard cycling conditions using PrimeTime® Gene Expression Master Mix
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Standard or fast cycling conditionsqPCR assays consisting of:• PrimeTime Gene Expression
Master Mix, reference dye• Varying amounts of cDNA 0.016–
50 ng
Efficient amplification under standard and fast cycling conditions
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Conclusions about the PrimeTime® Gene Expression Master Mix
• Works well for assays from IDT and other suppliers• Efficient amplification independent of the assay source
• Exhibits exceptional temperature and freeze-thaw stability• Reduced packaging resources and shipping costs• Ensured performance for high-throughput and overnight runs
• High efficiency when used for singleplex or multiplex reactions• Maximize use of rare samples and reduce reagent costs
• Performs well under standard or fast cycling conditions• Improved time from set-up to data
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