high efficiency qpcr with primetime® gene expression master mix from idt

Dr Erik Wendlandt Scientific Applications Specialist9/28/2016

High efficiency qPCR with PrimeTime® Gene Expression Master Mix

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′





• 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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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 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

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

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

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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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

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,




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The PrimeTime® Gene Expression Master Mix exhibits consistent performance after extended heat-stress

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

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

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,



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


Multiple freeze-thaw cycles do not affect the functionality of the master mix






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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

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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Primer-dimer analysis with the Oligo Analyzer® Tool

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PrimeTime® Multiplex Dye Selection Tool

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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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Master Mix, reference dye,• Varying amounts of cDNA 0.016–

50 ng


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Master Mix, reference dye• Varying amounts of cDNA 0.016–

50 ng

Efficient amplification under standard and fast cycling conditions

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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high efficiency qpcr with primetime® gene expression master mix from idt

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