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

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

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

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

• 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?

www.idtdna.com/order/Predesignedassay.aspx

The PrimeTime® Gene Expression Master Mix outperforms other commercially available master mixes

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

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

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

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

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

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

Consistent results for high throughput qPCR

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

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,

Consistent results under thermal stress

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

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

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

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

Considerations for multiplexing

• 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

Multiplexing with the PrimeTime® Gene Expression Master Mix(Overlay)

Singleplex

Yakima Yellow Cy5 Cy5.5

Singleplex

Multiplexing with the PrimeTime® Gene Expression Master Mix(Overlay)

Singleplex

Yakima Yellow Cy5 Cy5.5

Singleplex

Efficient amplification under singleplex and multiplex conditions

Primer-dimer analysis with the Oligo Analyzer® Tool

PrimeTime® Multiplex Dye Selection Tool

Efficiency of qPCR assays under fast and standard cycling conditions using PrimeTime® Gene Expression Master Mix

Standard or fast cycling conditionsqPCR assays consisting of:• PrimeTime Gene Expression

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

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

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

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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