PrimeTime qPCR Probe Assays

PrimeTime qPCR Probe Assays are offered in 3 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 or plate well. 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 96-well plate well
• Guaranteed performance for all predesigned assays
• Available in 5 dye–quencher combinations and 3 reaction scales
• MIQE compliant with all primer and probe sequences provided

Predesigned assays are available for human, mouse, and rat targets. These assays are designed using a proprietary algorithm. In addition to optimized oligo T_m (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.

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

PrimeTime assays are sensitive 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 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 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 Assays. The assays demonstrated high reproducibility between lots and across all 3 scales with negligible C_q 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 C_q 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.

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 C_q 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).

View all comparison assays »

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.

Aids for fluorophore and quencher selection

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.