PrimeTime® qPCR products for gene expression

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• Assays—probe- and primer-based• PrimeTime® qPCR Assays

(for human, mouse, and rat)• PrimeTime® Custom qPCR Assays

(for any sequence)

• Probes • ZEN™ or TAO™ Double-Quenched Probes• Wide selection of dyes

• Free design tools • PrimerQuest® Tool• RealTime Tool

Everything but your sample

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• Master mix• PrimeTime® Gene Expression Master Mix

(for probe-based assays)

• Controls• gBlocks® Gene Fragments

• Water and buffers• Nuclease-Free Water• IDTE Buffer

Probe-based and primer-only assaysPrimeTime® qPCR Assays

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Use PrimeTime® assays in probe-based and primer-only experiments

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• 5′ nuclease, probe-based assays(primers and probe set)

• Intercalating dye–based assays(primers only)

Advantages of PrimeTime® qPCR Assays Select from a database when studying gene expression in human, mouse and rat

• Primer and probe sequences provided with each purchase

• Better design delivers sensitive and high-efficiency assays• Based on up-to-date sequence information (exon boundaries, splice variants)• Designed to avoid SNPs

• Low price per reaction

• Stop waiting—assays shipped in 2–4 business days• PrimeTime® qPCR Assays are synthesized when the order is received

• Ensures you have assays designed with the most up-to-date sequence information

• All assay components undergo QC by mass spectrometry; probes are HPLC purified

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Cq values from PrimeTime® assays are equal to or approximately 1 Cq lower than from Competitor A assays

Better design delivers sensitive and efficient assays

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Mean  Cq  Value

Gene  RefSeq  Accession  Number

Comparison  of  Mean  Cq  Value

PrimeTime

Competitor  A

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IDT’s qPCR assay guarantee

Quality guaranteed

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“PrimeTime®  qPCR  Assays  will  perform  with  an  efficiency  between  90  and  110%  when  tested  over  a  5  log  dynamic  range  with  an  R2 fit  of  ≥0.99,  or  the  assay  will  be  replaced  at  no  additional  charge.”

PrimeTime® Custom qPCR AssaysNot working with human, mouse, or rat genomes?Have a unique sequence?

• Use our free PrimerQuest® Tool to design the best possible primer or probe targeting any sequence of interest

— Easily design: • With specific primer, probe, or amplicon criteria• Across a specified location• Around a fixed primer or probe location

— Software allows customization of ~45 parameters— Design algorithm includes multiple checks to reduce primer-dimer formation

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www.idtdna.com/primerquest

PrimeTime® Probes

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Choose your dye

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‡  For  reference  only.  Not  available  through  IDT:  VIC,  NED,  ABY,  PET,  JUN,  LIZ  dyes.

• PrimeTime® qPCR AssaysFAM, HEX™, TET™, Cy® 5

• PrimeTime Custom qPCR ProbesFAM, HEX, TET, Cy 5,JOE, Yakima Yellow®, Cy 3, Texas Red®, Tex 615™

• ATTO™ and AlexaFluor® dyes

Choose your quencher

11‡  For  reference  only.  Not  available  through  IDT.

• Recommended non-fluorescent quenchersZEN™ + Iowa Black® FQTAO™ + Iowa Black RQIowa Black RQ

Find the appropriate dyes for your instrument

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Use the next generation double-quenched probes

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Dyes Internal quencher 3′ quencherZEN™ Double-Quenched Probe: FAM,  TET™,  HEX™,  MAX™,  JOE ZEN™ IowaBlack® FQ

TAO™ Double-Quenched Probe: Cy®  5 TAO™ IowaBlack® RQ

ZEN™ Quencher: Low background delivers brighter probes

ZEN™ Double-Quenched Probes have lower background and increased sensitivity

(available with PrimeTime® qPCR Assays)

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PrimeTime® Gene Expression Master Mix

2X concentrated master mix for 2-step, probe-based assays

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PrimeTime® Gene Expression Master Mix:For probe-based assays

• 2-step RT-qPCR

• Direct analysis of DNA

• Singleplex or multiplex assays

• Fast or standard cycling conditions

• Overnight experiments (e.g., high-throughput experiments)

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PrimeTime® Gene Expression Master Mix provides convenience

• Use with your existing qPCR assays – Optimized to work with PrimeTime® qPCR Assays from IDT– Also works with:

• Custom primers/probe sets from IDT • qPCR assays from other manufacturers

• Use with your existing real-time PCR instrument– Use with standard or fast cycling conditions– Use with or without reference dye

• Incorporate  into  kits– Does  not  require  licensing  fees

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Consistent results from different lots support high throughput qPCR

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• Different batches of master mix generate reproducible results. The standard deviations of the Cq values for template levels >10 copies were <0.5.

• Consistent results, even after extended time at room temperature, means you can let your PCR experiments run overnight.

Part A: Consistent results between singleplex and duplex reactions—PrimeTime® qPCR Assays with Gene Expression Master Mix

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Amplification  plot:•  Singleplex,  black  curves•  Duplex,  red  curves

Assay  IDsHPRT:      Hs.PT.58v.45621572GUSB:    Hs.PT.58v.27737538

Part B: Consistent singleplex and duplex reactions–other commercial assay with the PrimeTime® Gene Expression Master Mix

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Amplification  plot:•  Singleplex,  black  curves•  Duplex,  red  curves

Thank you!

www.idtdna.com/PrimeTimewww.idtdna.com/qPCRprobeswww.idtdna.com/qPCRmastermix

www.idtdna.com/DECODED

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Supplemental slides:— GMP–licensing info— ZEN™ Double-Quenched Probe applications— PrimeTime® master mix data— Instrument dye calibration protocols— Additional qPCR products for genotyping

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No licensing fees

• PrimeTime® Gene Expression Master Mix• GMP oligos– Freedom™ dyes– Iowa Black® dark quenchers– ZEN™ and TAO™ quenchers

GMPinfo@idtdna.com

Licensing  fees  and  royalties  increase  as  sales  of  potential  kits  increase—using  license-­‐free  dyes,  quenchers,  and/or  master  mix  could  have  a  positive  effect  on  profitability.

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ZEN™ Double-Quenched Probes: Advantage in multiplex assays

“The ZEN Double-Quenched Probes have allowed us to continue adding probes to the already complex PRRSV assay mix to accommodate the changing viral landscape. This IDT product has helped us address the addition of new probes and primer sets to our assay very quickly.” —Dr Nelson, Founder of Tetracore

“In addition to providing us with the ZEN™ Double-Quenched Probes to help resolve our background issues, IDT has been invaluable, both from a quality and turnaround time perspective.”—Rolf Rauh, Scientist at Tetracore

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No bleed over signal with ZEN™Double-Quenched Probes

Bleed-over signal from poorly quenched probes

“Including the ZEN molecule as a second quencher has contributed significantly to increasing our signal-to-noise ratio.

When using dual-quenched probes, the negative drops emit less fluorescence than typically observed using standard probes with a single quenching moiety.

This provides a greater degree of separation between positive and negative clusters allowing for more precise counting of droplets in each population.” —Coren MilburySr. Scientist, Raindance Technologies

ZEN™ Double-Quenched Probes: For digital PCR

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“Performing MS-HRM or any kind of bisulfite sequencing reaction results in sequences with reduced cytosine content, and thus lower Tm.

This makes ZEN probes a great choice for these types of assays as the Tm of these longer probes can be kept high.

We switched to IDT PrimeTime qPCR Assays because the PrimeTime qPCR Assays work really well. They are much more cost efficient, and I really like how we can get them in different sizes so that when we are testing gene target assays, we can get just a small scale (Mini Assays), at a lower cost, to see if it would work for us. That was really appealing to us as a customer.”

Dr. Antoinette Perry,Trinity College , Dublin

ZEN™ Double-Quenched Probes: Maximum quenching with long probes for low-complexity sequences

Methylation studies

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Difference in Cq between PrimeTime® Gene Expression Master Mix and other master mixes

Same threshold and same y-axis

PrimeTime® Gene Expression MM – Alt MM

cDNA (ng) ∆Cq

gBlocks®

Fragments (copies)

∆Cq

50 –2.022 107 –2.463

10 –2.169 106 –2.675

2 –2.679 105 –3.098

0.4 –2.386 104 –3.096

0.08 –2.400 103 –3.169

102 –3.784

101 –3.862

cDN

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PrimeTime Gene Expression Master Mix Alternative master mix

gBlo

cks®

Frag

men

ts

PrimeTime HPRT Assay ID: s.PT.58v.45621572 27

• An overlay of the amplification plots for the PCRs using unheated or heated master mix shows no effect on the amplification curves.

PrimeTime® Gene Expression Master Mix—Exceptional stabilityReproducible amplification after heating the master mix at 55°C

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• Applied Biosystems® 7900 Fast Real-Time PCR System• HEX™ Dye• JOE Dye

• Applied Biosystems® 7300/7500/7500 Fast Real-Time PCR Systems• HEX™ Dye• JOE Dye• TET™ Dye

Download from www.idtdna.com/protocols

Dye calibration protocols

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Additional qPCR products

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• Genotyping• LNA PrimeTime® Probes and

Mini LNA PrimeTime® Probes• rhPrimers & RNase H2• MGB Eclipse® Probes*

*  Licensed  under  MGB  Eclipse  patents  for  use  by  end-­‐users  in  human  IVD  applications.For  use  as  a  component  in  the  Purchaser's  IVD  applications   only.MGB  Eclipse  is  a  registered  trademark  of  Elitech Group.

Overview slides:— PrimeTime® master mix— PrimeTime qPCR Assays— PrimerQuest® Tool

— SciTools (free, online software)— Online resources— Customer and technical support— Social media

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Make the most of your time in the lab.

www.idtdna.com/scitools.

• qPCR and PCR amplification

• Gene regulation and knockdown

• MicroRNA Analysis

• Gene design

• Sequencing

• Oligo dilution and resuspension

Our intuitive analysis and design tools and calculators are available for:

Begin designing at

Begin exploring at

Our Knowledge.YOUR RESOURCE.

www.idtdna.com/support.

Frequently Asked Questions

IDT Publications

Educational Resources

Technical Reports

User Guides & Protocols

Quick References

Video Library

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