Primer and probe premixed assays 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 assays for human, mouse, or rat for easy selection based on multiple criteria such as exon location and number of transcripts detected. 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.
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. 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
Predesigned assays are available for human, mouse, and rat targets. These assays are designed using a proprietary algorithm. In addition to optimized oligo 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.
We provide primer sequences with each order to assist with best practices in research reporting and reproducibility; specifically, sequence transparency:
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).
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).
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|
|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.
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 Cq variation (Figure 3).
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.
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 (NTC). 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.
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.
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 Universal Human Reference (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 Universal Human Reference cDNA are shown.
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.
We can provide custom formulation for PrimeTime qPCR Probe Assays. Contact Technical and Customer Support for assistance.
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.