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.
Available in various sizes, premixed, and shipped dried down. Typically ships within 2-4 business days.
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.
Available in various sizes, premixed, and shipped dried down. Typically ships within 5–9 business days.
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.
|5' dye||3' quencher||Mini||Standard||XL|
|FAM||ZEN™/Iowa Black™ FQ*||•||•||•|
|HEX||ZEN/Iowa Black FQ*||–||•||•|
|TET||ZEN/Iowa Black FQ*||–||•||•|
|Cy® 5||Iowa 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.
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.
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.
We provide primer sequences with each order to assist with best practices in research reporting:
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).
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. In this experiment, the data illustrates PCR efficiency and R2 values close to one across all dye/quenchers available for PrimeTime qPCR Probe Assays. All reactions were run with TaqMan® Gene Expression Master Mix (Thermo Fisher Scientific) under standard cycling conditions. The first four assays were run on the 7900 Fast Real-Time PCR System (Thermo Fisher Scientific) and the final assay (Cy5) was run on the LightCycler® 480 System (Roche).
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 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|
|Correlation coefficient (R²)||0.999||0.999||0.997||0.999||0.999|
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 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 shows greater than 90% efficiency and correlation coefficients greater than 0.99 for all tested qPCR master mixes.
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).
Figure 3. PrimeTime qPCR Probe Assays are consistent 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 Scientific). Each reaction contained 50 ng of cDNA. All assays were run in triplicate on the 7900 Real-Time PCR System (Thermo Fisher Scientific) using TaqMan® Gene Expression Master Mix (Thermo Fisher Scientific) 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.
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. A PrimeTime qPCR Probe Assay was analyzed by utilizing a plasmid 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 102.2% with a correlation coefficient of 0.9994.
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 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.
Twenty-five 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 the assays were comparison, 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 Scientific) and identical thresholds were set for all runs (Figures 5 and 6).
Figure 5. PrimeTime Assays have higher end-point fluorescence 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 Scientific). The reactions were run on the 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific) 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 mean Cq values for the same target. PrimeTime qPCR Assays were compared to equivalent Competitor A assays using five 4-fold dilutions of Universal Human Reference (UHR) cDNA and TaqMan Gene Expression Master Mix (Thermo Fisher Scientific). The reactions were run on the 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific) 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 Universal Human Reference cDNA are shown.
qPCR efficiency was compared using 25 PrimeTime qPCR Probe Assays and Competitor A assays (Figure 7). Again, the PrimeTime qPCR Assays showed 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 average 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 Scientific). The reactions were run on the 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific) 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.
No signal or amplification can result from a number of causes ranging from problems with the experimental design to inadvertently leaving out a necessary component from the reaction.
Here are some recommendations:
The cycle at which fluorescence from amplification exceeds the background fluorescence has been referred to as threshold cycle (Ct), crossing point (Cp), and take-off point (TOF) by different instrument manufacturers. But the term is now standardized by the MIQE guidelines as the quantification cycle( Cq). A lower Cq correlates with higher target expression in a sample.
Access the MIQE guidelines at:
Bustin SA, Benes V, et al. (2009) The MIQE Guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem, 55(4): 611−622. Updates: Bustin
SA, Beaulieu JF, et al. (2010) MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Mol Biol.11: 74–78, and Bustin SA, Benes V, et al. (2011) Primer sequence disclosure: A clarification of the MIQE Guidelines. Clin Chem, 57:919−921.
The stability of PrimeTime Gene Expression Master Mix makes it ideal for overnight PCR runs and high throughput experiments using robotic liquid handling systems.
Reliable results have been obtained when the qPCR was run directly after setup or after the reactions were maintained at room temperature for up to 72 hr.
Download the white paper, Ambient shipping of PrimeTime Gene Expression Master Mix for more data.
We provide a separate tube of reference dye so that you may use the PrimeTime Gene Expression Master Mix on reference dye-dependent or dye-independent instruments.
Also, the separate tube of reference dye provides a way to add a high or low dye concentration to the master mix, depending on the requirements of your instrument.
Yes, it uses an antibody-mediated, hot-start polymerase. During the initial denaturation step of qPCR cycling, polymerase activity is unblocked when the polymerase is released from the antibody.
This hot-start polymerase enhances reaction precision of the qPCR by avoiding non-specific amplification of DNA and primer dimer formation. It is therefore critical to follow the cycling conditions provided in the PrimeTime Gene Expression Master Mix Protocol.
The amount of reference dye that should be added to the PrimeTime Gene Expression Master Mix depends on which real-time PCR system you are using.
A table that details how much reference dye to add is provided in the PrimeTime Gene Expression Master Mix Protocol.
When performing qPCR it is ideal to have your probe Tm about 5−10 degrees higher than your primer Tm. The annealing temperature should be set 3−5 degrees lower than the lowest primer Tm.
Use this as a general guideline, but note that optimization may still be necessary.
We ship PrimeTime™ Gene Expression Master Mix, PrimeTime qPCR assays, and other oligonucleotide products at ambient temperature.
Different thermal cyclers have different requirements for dyes based on the excitation capability of the light source. Thus, different instruments have variable requirements for reference dye concentration.
Please refer to your instrument’s manual for information about these specific requirements.
Proofreading polymerases (Pfu, Diamond Taq®, etc) have been shown to remove 3' bases from PCR primers .
If you experience this problem, you can protect your primer from degradation by adding one or more phosphorothioate bonds to the 3' end of your primer.
Also make sure to add the enzyme last, after the addition of dNTPs, in your PCR to minimize the risk of this issue taking place.
1. Skerra A. Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity. Nucleic Acids Res. 1992;20(14):3551-3554.
To determine what dyes are compatible with your instrument, download our Real-time qPCR assay design guide or take a look at the PrimeTime™ Multiplex Dye Selection Tool.
Currently, the IDT ZEN quencher is available with FAM, HEX, TET, Yakima Yellow® (ELITech Group), JOE™ (Thermo Fisher Scientific), MAX™, and SUN™ fluorophores. Double-quenched probes have significantly lower background fluorescence, especially for longer probes.
The ZEN quencher is available on PrimeTime™ qPCR Probes and on the probes supplied with PrimeTime qPCR Probe Assays. For any special oligonucleotide requests that include the ZEN quencher but are not found on our website (non-catalog requests), please contact us.
For more information, see our DECODED™ article, Double-quenched probes increase qPCR sensitivity and precision.
*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.