PrimeTime® qPCR Assays

PrimeTime® qPCR 5' Nuclease Assays are probe-based assays—the gold standard for quantitative gene expression studies. Assays are available for various combinations of FAM, HEX, TET, and Cy5® dyes with ZEN™/Iowa Black FQ, TAMRA, and Iowa Black RQ quenchers. Choose from 3 reactions scales—Mini, Standard, and XL—according to the number of samples you plan to analyze.

PrimeTime® qPCR Primer Assays are provided without probe for intercalating dye experiments. Receive 5 nmoles each of forward and reverse primers mixed and delivered in a single tube or plate well, sufficient for 500 reactions (20 µL).

PrimeTime® qPCR 5' Nuclease Assays and PrimeTime® qPCR Primer Assays are available in tubes or 96-well plates. In compliance with MIQE Guidelines, all primer and probe sequences are provided with the order.

Easy, Custom qPCR Plate Design

  • Use the online design tool to create your own master plate; just cut, copy, paste, and fill in as you would with Excel.
  • Select from predesigned assays for human, mouse, or rat; or simply enter your own primer and probes sequences manually. Order assays with different dye–quencher combinations in the same plate.
  • All that is required is a minimum order of 24 assays or primer pairs per plate. Generate your own replicate plates for lower cost per reaction.
  • PrimeTime qPCR Primer Plates are shipped in just 3 days.  PrimeTime qPCR Assay Plates are shipped in less than 10 days.

PrimeTime® qPCR Assays

Order Assays in Tubes



Order Assays in Plates




Custom qPCR Design Tools (for other species and customized assay designs)

PrimerQuest® Tool

Highly customizable option for difficult PCR and qPCR designs.

RealTime PCR Tool


Design tool for species other than human, mouse, or rat.

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PrimeTime qPCR 5' Nuclease Assays are offered in three different sizes to meet any qPCR experimental need. In addition, for the Standard and XL sizes, selection of dye–quencher combination and primer-to-probe ratio can be specified to meet unique experimental demands. Assays consist of a forward primer, a reverse primer, and a qPCR probe all delivered in a single tube. Each assay is made to order with estimated shipping in 2–4 days from order receipt. Each oligonucleotide undergoes 100% QC by mass spectrometry and all QC results are provided free of charge to the customer on the IDT website.

  • Primers and probe mixed and delivered in a single tube or plate well
  • Online assay design tools provide guaranteed performance
  • Available in 5 dye–quencher combinations and 3 reaction scales
  • Choose ZEN™ Double-Quenched Probes in your Assay for superior performance compared to traditional dual-labeled probes.
  • MIQE Compliant: All primer and probe sequences are provided
  • Available in tubes or 96-well plates

PrimeTime® qPCR 5' Nuclease Assays in Tubes

No. of Reactions (20 µL) Price
(FAM-ZEN/Iowa Black FQ)
Price
(other dye-quencher combinations)1
Estimated Ship Date Probe (nmoles) Primers (nmoles)2
PrimeTime® Mini qPCR Assay100$75.00 USDNA2-4 business days0.51.0
PrimeTime™ Standard qPCR Assay500$120.00 USD$150.00 USD2-4 business days2.52.5-10
PrimeTime® XL qPCR Assay2500$350.00 USD$400.00 USD2-4 business days12.512.5-50

PrimeTime® qPCR 5' Nuclease Assays in 96-Well Plates



PrimeTime Assay Plates3Reactions (20 µL)Price per Assay (FAM/ZEN/Iowa Black FQ) Price per Assay (other dye-quencher combinations)1Estimated Ship DateProbe (nmoles) Primers (nmoles)3
PrimeTime™ Assay Plate Mini100$67.50 USDInquire 5-9 business days 0.51.0
PrimeTime™ Standard qPCR Assay500$108.00 USD$138.00 USD 5-9 business days 2.52.5 - 10
PrimeTime™ XL qPCR Assay2500$315.00 USD$365.00 USD 5-9 business days 12.52.5 - 50

1 See table below for available dye–quencher combinations.
2 The primer-to-probe ratio can be specified by the customer (except for PrimeTime Mini).
3 A minimum order of 24 assays is required per plate.

Available Dye and Quencher Combinations for PrimeTime® qPCR 5' Nuclease Assays

5' Dye 3' Quencher Mini Standard XL
FAM ZEN/Iowa Black FQ*
FAM TAMRA  
HEX ZEN/Iowa Black FQ*  
TET ZEN/Iowa Black FQ*  
Cy5 Iowa Black RQ  

*ZEN™/Iowa Black FQ is a Double-Quenched Probe which provides superior performance to traditional dual-labeled probes. For more information download the ZEN Double‑Quenched Probe Overview.

PrimeTime® qPCR 5' Nuclease Assays

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PrimeTime® qPCR Primer Assays are predesigned qPCR assays used to detect genes in the human, mouse, and rat transcriptomes. These primer sets are ideal for use with SYBR® Green, EvaGreen®, and other intercalating dyes, where no probe is needed. PrimeTime qPCR Primer Assays are available in Standard Size, premixed and normalized to 5 nmoles per primer, and are shipped dried down. Primer sequences are provided upon order, and the primers ship in 2–3 business days.

  • The same primer pairs found in the PrimeTime qPCR 5' Nuclease Assays
  • Forward and reverse primers mixed and delivered in a single tube or plate well
  • Ideal for use with SYBR® Green, EvaGreen, and other intercalating dyes, where no probe is needed
  • Available in tubes, 96-well plates, or Matrix plates

PrimeTime® qPCR Primer Assays in Tubes


No. of Reactions (20 µL)PriceEstimated Ship DateQuantity (nmoles)
PrimeTime qPCR Primers500$45.00 USD2-3 business days5

PrimeTime® qPCR Primer Assays in Plates


ProductReactions (20 µL)PriceEstimated Ship DateQuantity (nmoles)
PrimeTime™ Primer Plate 500 $40.00 USD 3 business days 5

PrimeTime qPCR Primer Assays 

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Step 1—The primers and probe hybridize in a sequence-dependent manner to the complementary DNA strand. Because the probe is intact, the fluorophore and quencher are in close proximity and the quencher absorbs fluorescence emitted by the fluorophore.

Step 2—The polymerase extends from the primers and begins DNA synthesis.

Step 3—The polymerase reaches the probe and the exonuclease activity of the polymerase cleaves the hybridized probe. As a result of cleavage, the fluorophore is separated from the quencher and fluoresces.

Step 4—The fluorescence is detected by the real time instrument.

These steps are repeated for each PCR cycle and allow detection of specific products. With intercalating dyes, such as SYBR® Green I, primer-dimers and non-specific products will also contribute to fluorescence. In contrast, the 5’ Nuclease assay is specific and fluorescence will only be detected for the DNA sequence to which the probe and primers hybridize.

Overview of 5′ Nuclease Assays

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Reporter Dyes

The correct reporter dye will depend on the type of instrument you are using and the compatibility of the dye with the instrument. Please see the Instrument Compatibility Table for a list of reporter dyes compatible with common instrumentation.

For multiplexing applications, it is recommended that reporters dyes with the least amount of spectral overlap be selected. For a complete list of IDT’s dyes and quenchers please see the Dye and Quencher Wavelength Figure and the Instrument Compatibility Table

Quenchers

Traditional dark quenchers absorb broadly and do not emit light which allows for the use of multiple reporter dyes with a given quencher. This characteristic allows for expanded options for multiplex assays. Dark quenchers simplify detection which makes them compatible with a broad range of image analysis instruments.

IDT has developed an internal ZEN quencher that enables to production of Double-Quenched Probes which have less background and more signal. The Double-Quenched Probes contain a 5’ FAM fluorophore, a 3’ IBFQ quencher, and an internal ZEN quencher. These Double-Quenched Probes are an improvement over traditional dual-labeled probes and have consistently low background, reduced Cq values, improved precision, and enable the use of longer probes for design in AT-rich regions. For more information download the ZEN Double-Quenched Probe Overview.

IDT supplies commonly used dark quenchers as well as the proprietary dark quenchers, Iowa Black FQ, Iowa Black RQ, and the newly developed internal ZEN quencher. TAMRA is also a quencher option for a FAM reporter dye. Related Information

Selecting the Correct Reporter Dye and Quencher

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Dye Calibration Protocols for Common Instruments

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IDT and Biogazelle are working together to provide users of PrimeTime® qPCR products a solution for qPCR data analysis.

Biogazelle is the real-time PCR data analysis company, built upon extensive experience in real-time PCR experiment design, assay development, and data-analysis. Groundbreaking publications on normalization of gene expression and data analysis by Biogazelle founders, Jo Vandesompele and Jan Hellemans, have been cited more than 3000 times.

qbase+ software from Biogazelle is a qPCR analysis package that allows direct import of data from qPCR instruments from a variety of manufacturers. The software is based on proven geNorm and qBase technology and enhanced with proprietary algorithms and time-saving features. These algorithms remove data errors, normalize data to one or more reference genes, and correct inter-run variation using inter-run calibrators. qbase+ software offers statistical tools for qPCR data analysis and graphical presentation tools for viewing analyzed data. It is the most powerful, flexible, and user-friendly real-time PCR data analysis software available. qbase+ is compatible with Windows, Mac, and Linux operating systems and meets MIQE guidelines.

Biogazelle offers a variety of other services to help investigators design and implement qPCR experiments, including educational materials and courses. For a complete overview of Biogazelle wet lab and data mining services, visit www.biogazelle.com.

Data Analysis Recommendations

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Suggested gene sets for some commonly studied pathways for human mouse and rat species are provided. Gene lists for additional signaling pathways (Notch, TLR , WNT, etc.), biochemical pathways, and genes that have been associated with certain cancers, can be found in the Extended Gene Sets List.

Copy and paste desired genes or gene sets into the search box of the PrimeTime Plates ordering tool to generate the required assays.

Human

Mouse

Rat

Extended Gene Sets List

Gene lists for additional signaling pathways (Notch, TLR , WNT, etc.), biochemical pathways, and genes that have been associated with certain cancers are available in Extended Human Target Gene List, a subset of data downloaded from the Broad Institute Molecular Signatures Database (MSigDB) [1], Version 3.1, September 2012.

References

  1. Subramanian A, Tamayo P, et al. (2005) Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA, 102(43):15545–15550.

Gene Sets for qPCR

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PrimeTime® Assays Excel with Fast-Cycling Protocols

Fast cycling allows for higher throughput and faster access to results. Unfortunately, researchers often have to sacrifice performance for speed. 6 PrimeTime qPCR Assays were tested using the Agilent Brilliant III Ultra Fast qPCR Master Mix, which allows run times as short as 45 minutes. These results were compared to 6 matched, inventoried assays from Competitor A.

  • No sacrifice in efficiency. All PrimeTime Assays maintained efficiencies of 90–100%. Two of the 6 assays from Competitor A had efficiency values <90%.
  • Greater sensitivity. PrimeTime qPCR Assays had lower Cq values compared to matched, inventoried assays from Competitor A by over 0.5 on average and ΔRn values that were almost 20% higher.

Increased Sensitivity

25 assays from Competitor A were compared to an equal number of IDT PrimeTime® qPCR 5' Nuclease Assays. The Competitor A assays consisted of 15 inventoried assays and 10 made-to-order assays. To ensure an accurate comparison was made, the PrimeTime Assays and Competitor A assays were selected to span the same exon boundary of each gene. The reactions were run with the Applied Biosystems Gene Expression Master Mix and identical thresholds were set for all runs (Figures 1 and 2).

View All Competitive Assays

Comparison of Assay Performance with WDR3 (NM_006784)

Figure 1. PrimeTime® Assays Are More Sensitive than Competitor A Assays. PrimeTime qPCR Assays were compared to equivalent Competitor A assays using five 4-fold dilutions of cDNA template and the Applied Biosystems TaqMan® Gene Expression Master Mix. The reactions were run on the ABI 7900HT Fast Real-Time PCR System with the following PCR cycling conditions: 2 min. 50°C; 10 min. 95°C; 45 x (15 sec. 95°C, 1 min. 60°C). Identical thresholds were set for all runs for comparison across assays. A comparison of the Competitor A WDR3 (NM_006784) assay and the equivalent IDT PrimeTime qPCR Assay is shown.

Comparison of Mean Cq Value

Figure 2. PrimeTime® qPCR Assays Have Consistently Lower Cq Values for the Same Target. PrimeTime qPCR Assays were compared to equivalent Competitor A assays using five 4-fold dilutions of UHR cDNA and Applied Biosystems TaqMan® Gene Expression Master Mix. The reactions were run on the ABI 7900HT Fast Real-Time PCR System with the following PCR cycling conditions: 2 min. 50°C; 10 min. 95°C; 45 x (15 sec. 95°C, 1 min. 60°C). Identical thresholds were set for all runs for comparison across assays. The mean Cq values from the 50 ng dilution of UHR cDNA are shown.

Higher qPCR Efficiency 

qPCR efficiency was measured using a comparison of 25 Competitor A and IDT assays for sensitivity (Figure 3). Again, the PrimeTime® qPCR Assays have a higher average qPCR efficiency than Competitor A assays. In addition, the overall distribution of qPCR efficiency was narrower and higher than that for Competitor A assays.

Comparison of qPCR Efficiency

Figure 3. PrimeTime® qPCR Assays Have Higher qPCR Efficiency and a Smaller Distribution Range than Competitor A Assays. PrimeTime qPCR Assays were compared to matched Competitor A assays using five 4-fold dilutions of cDNA and the Applied Biosystems TaqMan® Gene Expression Master Mix. The reactions were run on the Applied Biosystems 7900HT Fast Real-Time PCR System with the following PCR cycling conditions: 2 min 50°C; 10 min 95°C; 45 x (15 sec 95°C, 1 min 60°C). Identical thresholds were set for all runs for comparison across assays.

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Same High Efficiency With or Without Probe 

Panel A. Amplification with SYBR Green
Panel B. Amplification with Dual-Labeled Probe.
Figure 1. PrimeTime® Assays Yield the Same High Efficiency Whether Used With Intercalating Dyes or Probe. Amplification of 5 sequential 4-fold dilutions of cDNA using PrimeTime qPCR Primer Assays (with SYBR® Green dye) or the PrimeTime qPCR 5' Nuclease Assay (with dual-labeled probe) to human 3-oxoacid CoA transferase 1 (OXCT1) (NM_000436).
Figure 2. PrimeTime® qPCR Primer Assays Have Average Reaction Efficiency >90%. Sixty randomly selected PrimeTime qPCR Primer Assays and 15 PrimeTime qPCR Primer Assays for endogenous control genes used with Brilliant III Ultra Fast SYBR® Green qPCR Master Mix (Agilent) were analyzed over 5 sequential 4-fold dilutions (50–0.195 ng/reaction) of cDNA prepared from Universal Human Reference RNA (Agilent). Reactions were run on the 7900HT Fast Real-Time PCR System (Applied Biosystems) using PCR cycling conditions: 3 min 95°C; 45 x (5 sec 95°C, 15 sec 60°C). Average reaction efficiencies for the assays tested exceeded 98%.

PrimeTime® qPCR Primer Assays

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To demonstrate the performance of different dye–quencher combinations, IDT tested a dilution series and found robustness in PCR efficiency and R2 values across all dye–quencher combinations available. 

Dye–Quencher Combination FAM / Iowa Black FQ FAM / TAMRA HEX / Iowa Black FQ TET / Iowa Black FQ Cy5 / Iowa Black RQ
Amplification Curve
Standard Curve
Efficiency 95.7% 95.1% 94.7% 94.5% 98.0%
Correlation Coefficient (R²) >0.999 >0.999 >0.999 >0.999 >0.998

Demonstrated Assay Performance with Multiple Dye–Quencher Combinations. A plasmid dilution series of the CSK (c-src tyrosine kinase) Assay was used to test different dye–quencher combinations. The data illustrates robustness in PCR efficiency and R2 values close to 1 across all dye/quenchers available for PrimeTime® qPCR 5' Nuclease Assays. All reactions were run using Applied Biosystems Gene Expression Master Mix under standard cycling conditions. The first four assays were run on the Applied Biosystems 7900 Real-Time PCR Instrument and the final assay (Cy5) was run on the Roche LC480.

Dye-Quencher Combinations

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To determine the success of PrimeTime® qPCR Assays with commercially available master mixes, IDT tested five different master mixes over a dilution series of six orders of magnitude. The PrimeTime qPCR Assays demonstrated efficiency close to 100% across many commercially available master mixes as indicated below.
Product Qiagen QuantiTect Probe PCR Kit ABI TaqMan® Gene Expression Master Mix Bio-Rad iTaq™ Supermix with ROX Stratagene Brilliant II® QPCR Master mix Invitrogen Express qPCR SuperMix
Amplification Curve
Standard Curve
Efficiency 102.7% 102.5% 99.1% 100.7% 102.1%
Correlation Coefficient (R²) 0.999 0.999 0.997 0.999 0.999

Successful Amplification of PrimeTime® qPCR Assays with Various Commercial qPCR Master Mixes. A ten-fold dilution series over six orders of magnitude (1E7 to 100 copies) was created for the JAK2 transcript. The standard curves were generated by running the assay with the indicated commercial master mixes. The samples were run on the ABI 7900 under standard cycling conditions for 45 cycles. The data demonstrate greater than 90% efficiency and correlation coefficients greater than 0.99 for all tested qPCR master mixes.

Compatibility with Commercially Available Master Mixes

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For assay re-ordering, it is critical that manufacturing be reproducible from lot to lot. IDT tested five genes from two lots of PrimeTime® qPCR Assays with three replicates each. The lots were highly reproducible with very little CQ variation. Two lots of PrimeTime Mini qPCR Assays for five different genes were assessed.  

  Gene ID TNFRSF1A PDK1 JAK2 E2F1 TEC
Mini Replicate 1 28.9 24.6 27.5 22.9 29.1
Replicate 2 29.1 24.7 27.5 22.9 29.1
Replicate 3 28.8 24.6 27.5 22.9 29.1
Standard Replicate 1 29.0 24.6 27.3 22.8 29.6
Replicate 2 28.9 24.8 27.3 23.0 29.5
Replicate 3 28.9 24.6 27.5 22.9 29.6
XL Replicate 1 29.0 24.6 27.5 23.0 29.5
Replicate 2 28.9 24.6 27.5 23.0 29.4
Replicate 3 28.7 24.7 27.6 23.0 29.5
Amplification Curves

Lot-to-Lot Assay Reproducibility

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It is critical that assay performance remains consistent across the different assay scales. IDT tested the Mini, Standard, and XL PrimeTime® qPCR 5' Nuclease Assays and found reproducibility and precision across all three scales. This attribute simplifies the transition from discovery or validation applications to screening applications.

  Gene ID TNFRSF1A PDK1 JAK2 E2F1 TEC
Mini Replicate 1 28.9 24.6 27.5 22.9 29.1
Replicate 2 29.1 24.7 27.5 22.9 29.1
Replicate 3 28.8 24.6 27.5 22.9 29.1
Standard Replicate 1 29.0 24.6 27.3 22.8 29.6
Replicate 2 28.9 24.8 27.3 23.0 29.5
Replicate 3 28.9 24.6 27.5 22.9 29.6
XL Replicate 1 29.0 24.6 27.5 23.0 29.5
Replicate 2 28.9 24.6 27.5 23.0 29.4
Replicate 3 28.7 24.7 27.6 23.0 29.5
Amplification Curves

Performance of PrimeTime® qPCR 5' Nuclease Assays is Consistent Across Assay Scales. Reverse transcription of HeLa cell RNA was performed using oligo(dT) and random hexamers and SuperScript II (Invitrogen, Carlsbad, CA). Each reaction contained 50 ng of cDNA. All assays were run in triplicate on the Applied Biosystems 7900 Real-time PCR instrument using TaqMan Gene Expression Master Mix (Applied Biosystems) under standard cycling conditions for 45 cycles. The Cq values for three replicates are shown for each scale. Assays for five genes were formulated as PrimeTime Mini, Standard, and XL qPCR Assays.

Reproducibility Across Assay Scales

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To demonstrate the sensitivity of a PrimeTime® qPCR Assay, IDT tested a dilution over six orders of magnitude down to ten copies per reaction. All dilutions tested produced highly consistent results.

PrimeTime Dynamic Range
PrimeTime Dynamc Range Fig 1b

Dynamic Range (6 Logs) and 10 Copy Sensitivity. The PrimeTime® assay was analyzed by utilizing a plasmid dilution series, and a no template control. The data shown illustrates six logs of dynamic range and assay sensitivity down to 10 copies per reaction. The efficiency of the assay calculated from the standard curve is 102.2% with a correlation coefficient of 0.9994.

Dynamic Range and Sensitivity