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PrimeTime™ qPCR Probe Assays

Primer and probe premixed sequences for analyzing gene expression in any species using fluorescently labeled 5′ nuclease probes

PrimeTime qPCR Probe Assays consist of a primer pair and fluorescently labeled 5′ nuclease probe. Obtain predesigned sequences for human, mouse, or rat for easy selection based on multiple criteria such as exon location and number of transcripts. Create custom assays for any sequence from any species using the PrimerQuest™ Tool.

Ordering

  • Predesigned sequences will achieve 90% efficiency or better, or we will replace with an alternative design free of charge.
  • Select from a wide range of license-free dyes, including FAM, TET, SUN™, HEX, Cy® 5, Cy® 5.5, Yak™, Texas Red™, ATTO™ 647, and ATTO™ 425 dyes
  • Multiplex with confidence using double-quenched ZEN™ or TAO™ probe assays
  • Select a primer: probe ratio from 2:1 to 4:1 with no additional charge
  • Size and scale options available for digital PCR (dPCR) platforms
  • Receive primers and probe sequences with all orders

Have questions about PrimeTime qPCR Probe Assays?

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PrimeTime qPCR Probe Assays in tubes (1 probe/2 primers)

Available in various sizes, premixed, and shipped dried down. Typically ships within 2-4 business days.

5' Reporter Dye(s) / Emission (nm)3' QuencherMini (100-rxn)Standard (500-rxn)XL (2500-rxn)
ATTO 425
483
ZEN / Iowa Black™ FQ110,00 € EUR191,00 € EUR519,00 € EUR
FAM
520
ZEN / Iowa Black FQ83,00 € EUR150,00 € EUR420,00 € EUR
Black Hole Quencher 1110,00 € EUR191,00 € EUR519,00 € EUR
TAMRAN/A150,00 € EUR420,00 € EUR
TET
539
ZEN / Iowa Black FQN/A150,00 € EUR420,00 € EUR
YAK
551
ZEN / Iowa Black™ FQ174,00 € EUR297,00 € EUR796,00 € EUR
SUN
554
ZEN / Iowa Black™ FQ83,00 € EUR150,00 € EUR420,00 € EUR
Iowa Black FQ83,00 € EUR150,00 € EUR420,00 € EUR
HEX
555
ZEN / Iowa Black™ FQ83,00 € EUR150,00 € EUR420,00 € EUR
Black Hole Quencher 1110,00 € EUR191,00 € EUR519,00 € EUR
5' Texas Red®-X
617
Iowa Black RQ110,00 € EUR191,00 € EUR519,00 € EUR
Black Hole Quencher 2124,00 € EUR206,00 € EUR578,00 € EUR
Cy5
668
Iowa Black RQN/A191,00 € EUR519,00 € EUR
TAO / Iowa Black RQ110,00 € EUR191,00 € EUR519,00 € EUR
Black Hole Quencher 2124,00 € EUR206,00 € EUR578,00 € EUR
ATTO 647
662
Iowa Black RQ110,00 € EUR191,00 € EUR519,00 € EUR
Black Hole Quencher 2124,00 € EUR206,00 € EUR578,00 € EUR
Cy5.5
706
Black Hole Quencher 3174,00 € EUR297,00 € EUR796,00 € EUR

Probes/primers supplied in the following ratios: 0.5/1.0 nmol (Mini); 2.5/2.5–10 nmol (Standard); 12.5/12.5–50 nmol (XL). You may specify the primer-to-probe ratio (except for PrimeTime Mini).

* Predesigned assays are available for human, mouse, and rat targets.

PrimeTime qPCR Probe Assays in 96-well plates (1 probe/2 primers)

Available in various sizes, premixed, and shipped dried down. Typically ships within 59 business days.

5' Dye / Emission (nm)3' QuencherMini (100-rxn)Standard (500-rxn)XL (2500-rxn)
FAM 520ZEN / Iowa Black FQ*59,00 € EUR99,00 € EUR297,00 € EUR
TAMRAN/A128,00 € EUR338,00 € EUR
HEX 555ZEN / Iowa Black FQ*N/A128,00 € EUR338,00 € EUR
TET 539ZEN / Iowa Black FQ*N/A128,00 € EUR338,00 € EUR
Cy5 668Iowa Black RQN/A128,00 € EUR338,00 € EUR

Probes/primers supplied in the following ratios: 0.5/1.0 nmol (Mini); 2.5/2.5–10 nmol (Standard); 12.5/12.5–50 nmol (XL). You may specify the primer-to-probe ratio (except for PrimeTime Mini). A minimum order of 24 assays is required per plate.

Available dye and quencher combinations for PrimeTime qPCR 5′ Nuclease Assays in plate well format

5' dye3' quencherMiniStandardXL
FAMZEN™/Iowa Black FQ*
FAMTAMRA
HEXZEN/Iowa Black FQ*
TETZEN/Iowa Black FQ*
Cy® 5Iowa Black RQ

Key: • = available; – = not available

* ZEN/Iowa Black™ FQ is a Double-Quenched Probe, which provides superior performance compared to traditional single-quenched probes. For more information download the PrimeTime Custom qPCR Probes Flyer.

Product details

PrimeTime qPCR Probe Assays are offered in 3 different sizes to meet the needs of any qPCR experiment, which consist of a forward primer, a reverse primer, and a qPCR probe all delivered in a single tube or plate well. In addition, for the standard and XL sizes, dye–quencher combination and primer-to-probe ratio can be specified to meet your unique experimental demands. PrimeTime qPCR Probe Assays are made to order, and each oligonucleotide undergoes QC by mass spectrometry. All QC results are provided free of charge to you on the IDT website.

  • Primers and probe mixed and delivered in a single tube or 96-well plate well.
  • Predesigned sequences will achieve 90% efficiency or better, or we will replace them with an alternative design free of charge. Available in 18 dye–quencher combinations and 3 reaction scales.
  • MIQE compliant with all primer and probe sequences provided.

Predesigned sequences are available for human, mouse, and rat targets. These sequences are designed using a proprietary algorithm. In addition to optimized oligo melting temperature [Tm (based on composition, oligo length, etc.)], the bioinformatics calculations address avoidance of single nucleotide polymorphisms (SNPs; based on NCBI RefSeq releases) and off-target amplification, recognition of splice variants, and secondary structure predictions.

† With the exception of mixed base oligos, which could potentially represent multiple sequences and therefore cannot be accurately evaluated by ESI‑mass spectrometry.

Guarantee: Predesigned sequences will achieve 90% efficiency or better, or we will replace with an alternative design free of charge with the submission of supporting data. For more information, contact us.

Custom sequences can also be designed for other species, as well as human, mouse, and rat, using the PrimerQuest™ Tool. This tool may be used to design oligos for endpoint PCR, qPCR, and Sanger sequencing. Use our optimized preset design parameters for PCR and qPCR or customize parameters for your application. The PrimerQuest Tool is based on the Primer3 engine.

For commonly studied pathways in human, mouse, and rat species, we provide suggested gene sets (see Resources section below) that can be used with our PrimeTime plate ordering system.

Primer sequences are provided

We provide primer sequences with each order to assist with best practices in research reporting:

  • Boosts publication credibility—you can publish you research according to the MIQE guidelines
  • Helps identify and avoid SNPs
  • Facilitates design of multiplex experiments
  • Assists with data interpretation and troubleshooting
  • Allows you to valid your transcripts

Product data

PrimeTime qPCR Probe Assays provide reliable results

To demonstrate the performance of different dye-quencher combinations, we tested a dilution series to evaluate PCR efficiency and R2 values across all dye-quencher combinations available (Figure 1).

Dye-quencher combination FAM/ZEN/Iowa Black FQ Sun/ZEN/Iowa Black FQHEX/ZEN/Iowa Black FQ TET/ZEN/Iowa Black FQ Cy5/TAO/Iowa Black RQ
Amplification curve Amplification Curve - FAM / Iowa Black FQ Amplification Curve - FAM / TAMRA Amplification Curve - HEX / Iowa Black FQ Amplification Curve - TET / Iowa Black FQ Amplification Curve - Cy5/Iowa Black RQ
Standard curve Standard Curve - FAM / Iowa Black FQ Standard Curve - FAM / TAMRA Standard Curve - HEX / Iowa Black FQ Standard Curve - TET / Iowa Black FQ Standard Curve - Cy5/Iowa Black RQ
Efficiency 97.499.7% 98.4% 97.2% 95.0%
Correlation coefficient (R²) 0.9970.9980.9970.998 0.999

Figure 1. Demonstrated assay performance with multiple dye–quencher combinations. A gBlocks Gene Fragment dilution series of the HPRT gene was used to test different dye-quencher combinations. The data show PCR efficiency and R2 values close to 1 across all dye/quenchers available for PrimeTime qPCR Assays. All reactions were run using IDT’s PrimeTime Gene Expression Master Mix under standard two step cycling conditions 95°C for 3 min, (95 °C for 15 sec + 60°C for 1 min) x 40 on the QuantStudio™ 7 Flex (Thermo Fisher Scientific) 386 well format. Reactions were 10 µL in volume and contained 500 nM primers and 250 nM probe. The PrimeTime Gene Expression Master Mix was used with low ROX reference dye.

 

Compatibility with commercially available master mixes

To determine the success of PrimeTime qPCR Probe Assays with commercially available master mixes, we tested 5 different master mixes over a dilution series of 6 orders of magnitude (Figure 2). The PrimeTime qPCR Probe Assays had efficiency close to 100% across many commercially available master mixes. We have also developed the PrimeTime Gene Expression Master Mix for use with PrimeTime probe-based assays in two-step RT-qPCR.

Product Qiagen QuantiNova Probe PCR Kit Thermo Fisher Scientific  TaqMan Fast Advanced Bio-Rad Sso Advanced Aligent Stratagene Brilliant II® Takara Premix Ex Taq IDT Gene Expression Master Mix
Amplification curve Amplification Curve - Qiagen QuantiTect Probe PCR KitAmplification Curve - AB TaqMan® Gene Expression Master MixAmplification Curve - Bio-Rad iTaq™ Supermix with ROXAmplification Curve - Stratagene Brilliant II® QPCR Master mixAmplification Curve - Invitrogen Express qPCR SuperMixAmp-Curve-IDT-Gene-Expression-Master-Mix
Standard curve Standard Curve - Qiagen QuantiTect Probe PCR KitStandard Curve - AB TaqMan® Gene Expression Master MixStandard Curve - Bio-Rad iTaq™ Supermix with ROXStandard Curve - Stratagene Brilliant II® QPCR Master mixStd Curve - Takara Premix Ex Taq Std Curve - IDT Gene Expression Master Mix
Efficiency 102.1% 98.6% 99.8% 98.0% 101.8% 99.1%
Correlation coefficient (R²) 0.9980.998 0.996 0.996 0.999 0.998

Figure 2. Successful amplification of PrimeTime qPCR Probe Assays with various commercial qPCR master mixes. A 10-fold dilution series over 6 orders of magnitude (1 x 106 to 1 x 101 copies) was created for the HPRT1 transcript. The standard curves were generated by running the assay with the indicated commercial master mixes. The samples were run on a Thermo Fisher Scientific QuantStudio™ 7 Flex instrument under standard cycling conditions for 40 cycles. The data shows greater than 90% efficiency and correlation coefficients greater than 0.99 for all tested qPCR master mixes.

 

Low variability between assay lots and across assay scales

PrimeTime qPCR Probe Assays have low variability from lot to lot and across scales, which supports your research needs for re-ordering consistent assays and for transitioning from discovery or validation applications to screening. We tested 5 genes from 2 lots each of mini, standard, and XL PrimeTime qPCR Probe Assays. The assays showed consistency between lots and across all 3 scales with negligible Cq variation (Figure 3).

Gene ID
 HPRT1 PGK1 RPLP0 UBC WDR3
Mini Replicate 1 27.123.721.121.826.7
Replicate 2 27.123.721.121.826.6
Standard Replicate 1 27.223.721.121.626.5
Replicate 2 26.923.521.121.626.4
XL Replicate 1 27.123.521.021.326.4
Replicate 2 27.023.521.121.426.3
Amplification curves Amplification Curve - TNFRSF1AAmplification Curve - PDK1Amplification Curve - JAK2Amplification Curve - E2F1Amplification Curve - TEC

Figure 3. PrimeTime qPCR Probe Assays are consistent from lot to lot and across scales. Reverse transcription of qPCR Human Reference total RNA (Agilent) was performed using oligo(dT), random hexamers, and SuperScript® II (Thermo Fisher Scientific). Each qPCR reaction contained 50 ng of cDNA. All assays were run in duplicate on the CFX384 Touch Real-Time PCR system (BioRad) for 45 cycles using the PrimeTime Gene Expression Master Mix. The Cq values for the 2 replicates are shown. Assays for 5 genes were formulated as PrimeTime Mini, Standard, and XL qPCR Assays.

 

Dynamic range down to 10 copies

To show the dynamic range for PrimeTime qPCR Probe Assays, we tested a dilution over 6 orders of magnitude down to 10 copies per reaction (Figure 4). All dilutions tested produced highly consistent results.

Figure 4. Dynamic range (6 logs) down to 10-copies. An HPRT PrimeTime qPCR Probe Assay was analyzed by utilizing a gBlock Gene Fragments dilution series and a no template control (NTC). The data shown illustrates 6 logs of dynamic range and assay down to 10 copies per reaction. The efficiency of the assay calculated from the standard curve is 97.6% with a correlation coefficient of 0.9991.

PrimeTime qPCR Probe 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. PrimeTime qPCR Probe Assays were tested using the PrimeTime Gene Expression Master Mix, which allows run times as short as 45 minutes. These results were compared to 30 matched, inventoried assays from a competitor.

  • Greater low copy input limit─twelve out of the sixteen PrimeTime qPCR Probe Assays had lower Cq values compared to matched, inventoried assays from a competitor.
  • No sacrifice in efficiency─all PrimeTime Assays maintained efficiencies of 90–105%.

Lower mean Cq values

Sixteen assays from a competitor were compared to an equal number of PrimeTime qPCR 5' Nuclease Assays. To ensure the assays were comparable, the PrimeTime Assays and Competitor assays were selected to span the same exon boundary of each gene. The reactions were run with the PrimeTime Gene Expression Master Mix and identical thresholds were set for all runs (Figures 5).

Figure 5. PrimeTime qPCR Assays have consistently lower mean Cq values for the same target. PrimeTime qPCR Assays were compared to matched Competitor assays using five, 4-fold dilutions of cDNA and the PrimeTime Gene Expression Master Mix. The reactions were run on the CFX384 Touch Real-Time PCR system (BioRad) for 45 cycles 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.

Higher qPCR efficiency

qPCR efficiency was compared using 30 PrimeTime qPCR Probe Assays and Competitor A assays (Figure 6). Again, the PrimeTime qPCR Assays showed a higher average qPCR efficiency than Competitor assays. In addition, the overall distribution of qPCR efficiency was narrower and higher than that for Competitor assays.

Figure 6. PrimeTime qPCR Assays have higher average qPCR efficiency and a smaller distribution range than Competitor assays. PrimeTime qPCR Assays were compared to matched Competitor assays using five, 4-fold dilutions of cDNA and the PrimeTime Gene Expression Master Mix. The reactions were run on the CFX384 Touch Real-Time PCR Detection System (BioRad) 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.

Resources

Gene sets for common pathways

Frequently asked questions

RUO22-1558_003