qPCR Guide

PrimeTime® qPCR Probes

  • Dual-labeled probes are available with a wide variety of dyes and quenchers.
  • Express probes are ready for shipment in a single working day.
  • Ultra-small-scale Mini Probes allow you to try out a new probe or screen the expression levels of many genes.
  • Choose ZEN Double-Quenched Probes for superior performance compared to traditional dual-labeled probes.

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PrimeTime qPCR DNA probes are non-extendable oligonucleotides labeled with a 5’ fluorescent reporter and a 3’ quencher dye. For more information on dyes and quenchers, please visit the support tab. The probes are licensed for use in the 5’ Nuclease Real-Time PCR assay (with companion dye and quencher licenses). PrimeTime qPCR probes are available with a variety of reporter/quencher combinations and synthesis scales.

The prices and estimated turnaround time for these probes will depend on the degree of complexity. Probes at the Standard Level will ship in approximately 3-5 business days while probes at the Complex Level will ship in approximately 4-6 business days. The minimum guarantees are listed for probes that are 15-35 bases in length.

Please inquire about yield and price for probes 36-45 bases in length If you require qPCR probes for an application other than the 5’ Nuclease Real-Time PCR assay, please use the DLP builder.

PrimeTime qPCR Probes - Standard Level

5' Reporter Dye(s)QuencherSynthesis Scale / Minimum Guarantee
100 nmole / 15 nmoles250 nmole / 25 nmoles1 umole / 50 nmoles
FAMZEN / Iowa Black FQ *$195.00 USD$275.00 USD$415.00 USD
5' Reporter Dye(s)QuencherSynthesis Scale / Minimum Guarantee
100 nmole / 10 nmoles250 nmole / 25 nmoles1 umole / 50 nmoles
FAMTAMRA$215.00 USD$300.00 USD$460.00 USD
Black Hole Quencher 1$215.00 USD$300.00 USD$460.00 USD
HEXZEN / Iowa Black FQ *$245.00 USD$350.00 USD$520.00 USD
Black Hole Quencher 1$265.00 USD$380.00 USD$575.00 USD
TETZEN / Iowa Black FQ *$245.00 USD$350.00 USD$520.00 USD
Black Hole Quencher 1$265.00 USD$380.00 USD$575.00 USD

*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.

The estimated turnaround time for Standard Level Probes is 3–5 business days.

PrimeTime qPCR Probes - Complex Level

5' Reporter Dye(s)QuencherSynthesis Scale / Minimum Guarantee
100 nmole / 2 nmoles250 nmole / 8 nmoles1 umole / 20 nmoles
FAMTAMRA NHS EsterN/A$450.00 USD$640.00 USD
MAX NHSZEN / Iowa Black FQ *$445.00 USD$515.00 USD$765.00 USD
Black Hole Quencher 1$445.00 USD$515.00 USD$765.00 USD
Cy3Iowa Black RQ$225.00 USD$260.00 USD$475.00 USD
Black Hole Quencher 2$345.00 USD$395.00 USD$600.00 USD
TEX 615Iowa Black RQ$315.00 USD$365.00 USD$665.00 USD
Black Hole Quencher 2$445.00 USD$515.00 USD$765.00 USD
Cy5Iowa Black RQ$225.00 USD$260.00 USD$475.00 USD
Black Hole Quencher 2$345.00 USD$395.00 USD$600.00 USD
TYE 563Iowa Black RQ$315.00 USD$365.00 USD$665.00 USD
Black Hole Quencher 2$445.00 USD$515.00 USD$765.00 USD
TYE 665Iowa Black RQ$315.00 USD$365.00 USD$665.00 USD
Black Hole Quencher 2$445.00 USD$515.00 USD$765.00 USD
JOE NHS EsterZEN / Iowa Black FQ *N/A$350.00 USD$520.00 USD
Black Hole Quencher 1N/A$380.00 USD$575.00 USD
TAMRA NHS EsterIowa Black RQN/A$345.00 USD$515.00 USD
Black Hole Quencher 2N/A$455.00 USD$695.00 USD
ROX NHS EsterIowa Black RQN/A$345.00 USD$515.00 USD
Black Hole Quencher 2N/A$455.00 USD$695.00 USD
Texas Red-X NHS EsterIowa Black RQN/A$345.00 USD$515.00 USD
Black Hole Quencher 2N/A$455.00 USD$695.00 USD

The estimated turnaround time for Complex Level Probes is 4-6 business days.

PrimeTime qPCR Probes

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The PrimeTime Mini qPCR probes are probes available on a small scale. The smaller scale and lower price makes them ideal for trying out a new probe, for screening the expression levels of many genes, or for testing the conversion to FAM/IBFQ and FAM/ZEN/IBFQ from other FAM-related quenchers. Mini probes are offered with a 5’ FAM and the option of a 3’ IBFQ quencher alone or in combination with the internal ZEN quencher. The estimated turnaround time is 3-5 days.

PrimeTime Mini qPCR Probes


5' Reporter Dye(s)Quencher(s)Delivery Amount
0.5 nmoles
FAMZEN / Iowa Black FQ *$65.00 USD

*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.

The turnaround time from order to ship for PrimeTime Mini qPCR Probes is 3-5 business days.

PrimeTime Mini qPCR Probes

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Express qPCR probes allow researchers the opportunity to have their qPCR probes ship in just one working day! All oligos are HPLC purified and quality control checked by mass spectrometry and the trace is available free on our website. Due to the fast turnaround time, these probes are limited to select dye-quencher combinations and synthesis scales.

All Express orders must be placed online by 2:00pm ET. The orders will be shipped with next day priority for delivery by 10:30am. All Express probes must be 18-35 bases, will be HPLC purified and QC verified by mass spectrometry. Probes will be shipped lyophilized and will have a minimum guarantee of 5 nmoles. Additional shipping and handling fees for expedited service apply.

PrimeTime Express qPCR Probes

5' Reporter Dye(s)Quencher(s)Synthesis Scale / Minimum Guarantee
100nm / 5 nmoles
FAMZEN / Iowa Black FQ *$249.00 USD
Iowa Black$229.00 USD
Black Hole Quencher-1$270.00 USD

*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.

The turnaround time from order to ship for Express Dual Labeled Probes is 1 business day.

Express qPCR Probes

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In step one, 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.

In step two, the polymerase extends from the primers and begins DNA synthesis.

In step three, 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. In step four, this fluorescence is detected by the real time instrument. These steps are repeated for each PCR cycle and allow detection of specific products. With intercalation 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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Double-Quenched Probes

IDT has developed a new internal ZEN quencher which enables the production of Double-Quenched Probes with less background and increased signal! Double-Quenched Probes contain a 5’ FAM fluorophore, a 3’ IBFQ quencher, and an internal ZEN quencher. While traditional probes have around 20-30 bases between the fluorophore and the quencher, the internal ZEN quencher decreases that length to only 9 bases. This shortened distance, particularly when combined with the traditional 3’ end quencher, leads to a much more thorough quenching with much less background and enables the use of much longer probes for designing in AT-rich target regions. In addition to the significantly decreased background, the Double-Quenched Probes also have consistently reduced Cq values and improved precision when compared to traditional probes. Use of the Double-Quenched Probes can allow users to experience both increased sensitivity and precision in their qPCR experiments.

For more information download the ZEN Double-Quenched Probe Overview.

ZEN Double-Quenched Probes™

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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.

Figure 1. 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

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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.
ProductQiagen QuantiTect Probe PCR KitABI TaqMan® Gene Expression Master MixBio-Rad iTaq™ Supermix with ROXStratagene Brilliant II® QPCR Master mixInvitrogen Express qPCR SuperMix
Amplification Curve
Standard Curve
Efficiency102.7%102.5%99.1%100.7%102.1%
Correlation Coefficient (R²)0.9990.9990.9970.9990.999

Figure 2. 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.

PrimeTime Reliability 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.

Fig. 3A

  Gene ID E2F1 JAK2 PDK1 TEC TNFRSF1A
Lot 1 Replicate 1 22.5 27.4 24.3 29.3 28.8
Replicate 2 22.5 27.2 24.4 29.4 28.9
Replicate 3 22.5 27.4 24.2 29.1 29.0
Lot 2 Replicate 1 22.6 27.4 24.5 29.2 29.0
Replicate 2 22.5 27.4 24.4 29.5 28.9
Replicate 3 22.6 27.3 24.4 29.4 29.0
Amplification Curves

Lot to Lot Assay Reproducibility

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Aside from lot to lot variation, it is critical that the performance of the assay remain consistent across scales. IDT tested the Mini, Standard and XL PrimeTime® qPCR Assays and found reproducibility and precision across all three scales. This attribute simplifies transition from discovery or validation applications to screening applications.

Fig. 3B

  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

Figure 3. Each Reaction Contained 50 ng of HeLa cell cDNA and the ABI Taqman Gene Expression Master Mix. The cDNA was made with oligo dT and random hexamers using SuperScript II (Invitrogen, Carlsbad, CA). All assays were run in triplicate using the ABI 7900 Real-time PCR instrument under standard cycling conditions for 45 cycles. Each table lists CQ values with three replicates each. (A) Two lots of PrimeTime Mini qPCR Assays were manufactured for five different assays. (B) Assays for five genes were formulated as a PrimeTime Mini, Standard, and XL qPCR Assays.

Reproducibility Across Assay Scales

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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

Figure 4. 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 one across all dye/quenchers available for PrimeTime Assays. All reactions were run with ABI Gene Expression Master Mix under standard cycling conditions. The first four assays were run on the ABI 7900 and the final assay (Cy5) was run on the Roche LC480.

Dye-Quencher Combinations

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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 between 90 and 100%. Two of the 6 Competitor A assays 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.
Comparison of NM_014742 Assay Performance Comparison of NM_014630 Assay Performance Comparison of NM_014367 Assay Performance Comparison of NM_014155 Assay Performance Comparison of NM_014109 Assay Performance Comparison of NM_012204 Assay Performance Comparison of NM_007024 Assay Performance Comparison of NM_006784 Assay Performance Comparison of NM_006782 Assay Performance Comparison of NM_006500 Assay Performance Comparison of NM_006325 Assay Performance Comparison of NM_005911 Assay Performance Comparison of NM_005768 Assay Performance Comparison of NM_005385 Assay Performance Comparison of NM_005188 Assay Performance Comparison of NM_004343 Assay Performance Comparison of NM_004308 Assay Performance Comparison of NM_003922 Assay Performance Comparison of NM_003596 Assay Performance Comparison of NM_003105 Assay Performance Comparison of NM_002581 Assay Performance Comparison of NM_001855 Assay Performance Comparison of NM_001182 Assay Performance Comparison of NM_000671 Assay Performance Comparison of NM_000362 Assay Performance
  WDR3 TIMP3 HERC1
Mean Cq (50 ng) PrimeTime® 22.9 23.9 25.1
Competitor A 24.0 24.1 26.2
Efficiency PrimeTime® 97.0% 95.0% 95.9%
Competitor A 96.4% 91.7% 86.8%
Endpoint (ΔRn) PrimeTime® 10.9 8.9 7.9
Competitor A 7.2 6.1 7.0

Figure 5. PrimeTime® qPCR Assays Maintain Efficiency Under Fast-Cycling Conditions. 25 PrimeTime qPCR Assays were compared to equivalent Competitor A assays (consisting of 15 inventoried pre-designed assays and 10 made-to-order pre-designed 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. Reactions included five 4-fold dilutions cDNA template and the Applied Biosystems TaqMan® Gene Expression Master Mix, and 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. 


Increased Sensitivity

25 assays from Competitor A were compared to an equal number of IDT PrimeTime® qPCR 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 6 and 7).

Comparison of Assay Performance with WDR3 (NM_006784)

Figure 6. 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 7. 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 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. 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 8). 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 8. 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 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.



PrimeTime qPCR Assay Competitor Comparison