gene_expression

PrimeTime® qPCR Probe Assays

Primer and probe premixed assays for analyzing gene expression in any species using 5′ nuclease fluorescent probes

PrimeTime qPCR Probe Assays consist of a primer pair and 5′ nuclease probe. Predesigned assays for human, mouse, or rat are available for easy selection based on multiple criteria such as exon location and number of transcripts detected. Custom assays may be created for any sequence from any species using the PrimerQuest® tool.

  • Achieve >90% efficiency over 4 or more orders of magnitude with our predesigned assays, guaranteed
  • Multiplex with confidence with double-quenched probe assays that have the lowest background in the industry
  • Adjust your primer probe ratio from 2:1 to 4:1 with no additional charge
  • Receive primers and probe sequences with all orders

Ordering

PrimeTime qPCR Probe Assays in tubes

Available in various sizes, premixed, and shipped dried down.

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$80.00 USDNA2-4 business days0.51.0
PrimeTime Standard qPCR Assay500$130.00 USD$163.00 USD2-4 business days2.52.5-10
PrimeTime® XL qPCR Assay2500$380.00 USD$435.00 USD2-4 business days12.512.5-50

1 See Product details section for available dye–quencher combinations.

2 You may specify the primer-to-probe ratio (except for PrimeTime Mini).

3 Available for human, mouse, and rat targets.

PrimeTime qPCR Probe Assays in 96-well plates

Available in various sizes, premixed, and shipped dried down.


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 Product details section for available dye–quencher combinations.

2 You can specify the primer-to-probe ratio (except for PrimeTime Mini).

3 A minimum order of 24 assays is required per plate.

PrimeTime qPCR 5′ nuclease assays are offered in three different sizes to meet the needs of any qPCR experiment. Assays consist of a forward primer, a reverse primer, and a qPCR probe all delivered in a single tube. In addition, for the standard and XL sizes, dye–quencher combination and primer-to-probe ratio can be specified to meet unique experimental demands. 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 you on the IDT website.

  • Primers and probe mixed and delivered in a single tube or plate well
  • Guaranteed performance for all predesigned assays
  • Available in 5 dye–quencher combinations and 3 reaction scales
  • Choose ZEN Double-Quenched Probes for 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

Predesigned assays are available for human, mouse, and rat targets. These assays are designed using a proprietary algorithm. In addition to optimized oligo Tm (base composition, oligo length, etc.), the bioinformatic calculations account for factors, such as SNPs (based on current NCBI RefSeq releases), cross-react searches to avoid off-target amplification, recognition of splice variants, and secondary structure predictions.

Guarantee: Predesigned assays are guaranteed to perform with PCR efficiencies of 90–110% and R2 >0.99, or the assay will be replaced with another at no additional charge. Supporting data must be submitted to applicationsupport@idtdna.com.

Custom 5′ nuclease assays can also be designed for other species 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 Primer 3 engine.

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

For help with assay design, contact applicationsupport@idtdna.com.

Available dye and quencher combinations for PrimeTime qPCR 5′ Nuclease Assays

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.

Primer sequences are provided

We provide primer sequences with each order to assist with best practices in research reporting and reproducibility. Specifically, sequence transparency

  • 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 validation of new transcripts

Overview of 5′ nuclease assays

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. When using 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.

Selecting the correct reporter dye and quencher

  • 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, we recommend using reporter dyes with the least amount of spectral overlap. For assistance in selecting multiplex dye combinations, use our Multiplexing Dye Selection Tool, which helps you select dyes based on what instrument you have. As an additional reference, for a list of selected IDT 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. We have developed an internal ZEN quencher that enables the 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 C q values, improved precision, and enable the use of longer probes for design in AT-rich regions. For more information download the PrimeTime Custom qPCR Probes flyer. We supply commonly used dark quenchers as well as the proprietary dark quenchers, Iowa Black FQ, Iowa Black RQ, and the internal ZEN Quencher. TAMRA is also a quencher option for a FAM reporter dye.

See Resources section for imformation on dye and quencher wavelengths and instrument compatibility.

PrimeTime assays are sensitve and provide reliable results

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

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 1. 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 TaqMan® Gene Expression Master Mix (Thermo Fisher) under standard cycling conditions. The first four assays were run on the 7900 Fast Real-Time PCR System (Thermo Fisher) and the final assay (Cy5) was run on the LightCycler® 480 System (Roche).

Compatibility with commercially available master mixes

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

Product Qiagen QuantiTect Probe PCR Kit AB 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

Figure 2. Successful amplification of PrimeTime qPCR Assays with various commercial qPCR master mixes. A 10-fold dilution series over 6orders of magnitude (1 x 107 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 an Applied Biosystems 7900 instrument 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.

Reproducibility between assay lots and across assay scales

Assays must be reproducible from lot to lot and across scales, for example, to meet research needs for re-ordering 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 5′ Nuclease Assays. The assays demonstrated high reproducibility between lots and across all 3 scales with negligible Cq variation (Figure 3).

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. PrimeTime qPCR assays are reproducible from lot to lot and across scales. Reverse transcription of HeLa cell RNA was performed using oligo(dT) and random hexamers and SuperScript® II (Thermo Fisher). Each reaction contained 50 ng of cDNA. All assays were run in triplicate on the 7900 Real-Time PCR System (Thermo Fisher) using TaqMan® Gene Expression Master Mix (Thermo Fisher) under standard cycling conditions for 45 cycles. The Cq values for 3 replicates are shown. Assays for 5 genes were formulated as PrimeTime Mini, Standard, and XL qPCR Assays.

Dynamic range and sensitivity

To demonstrate the sensitivity of PrimeTime qPCR 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) and 10-copy sensitivity. A PrimeTime assay was analyzed by utilizing a plasmid dilution series and a no-template control. The data shown illustrates 6 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.

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. 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.

  • 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.
  • No sacrifice in efficiency: All PrimeTime Assays maintained efficiencies of 90–100%. Two of the 6 assays from Competitor A had efficiency values <90%.

Increased sensitivity

25 assays from Competitor A were compared to an equal number of 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 TaqMan Gene Expression Master Mix (Thermo Fisher) and identical thresholds were set for all runs (Figures 5 and 6).

Figure 5. 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 TaqMan Gene Expression Master Mix (Thermo Fisher). The reactions were run on the 7900HT Fast Real-Time PCR System (Thermo Fisher) 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 PrimeTime qPCR Assay are shown.

Figure 6. 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 TaqMan Gene Expression Master Mix (Thermo Fisher). The reactions were run on the 7900HT Fast Real-Time PCR System (Thermo Fisher) 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. Mean Cq values from the 50 ng dilution of UHR cDNA are shown.

Higher qPCR efficiency

qPCR efficiency was compared using 25 Competitor A and PrimeTime assays (Figure 7). Again, the PrimeTime qPCR Assays demonstrated 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.

Figure 7. 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 TaqMan Gene Expression Master Mix (Thermo Fisher). The reactions were run on the 7900HT Fast Real-Time PCR System (Thermo Fisher) 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.

Gene sets for common pathways

Formulation

We can provide custom formulation for PrimeTime qPCR Probe Assays. Contact Technical and Customer Support for assistance.

GMP and OEM

If you require qPCR probes and primers that are approved for use in molecular diagnostic applications, or if you are interested in our OEM services, please click here.