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| Documentation and Support |
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| | Validated design tool for target sequences contained within the RefSeq database. Guaranteed to provide qPCR efficiency >90% when run under default settings. | | | | | Custom assay design tool. Assays for target sequences that are not part of the RefSeq database can be entered. Assay locations, reaction and design parameters can be customized individually. |
| | Primers and probes mixed together and shipped in a single tube.
*Now with added flexibility! Select from a number of different dye-quencher combinations and specify your primer to probe ratio. | | | | | Select from a wide variety of dyes, quenchers and scales | | | | | Dual Labeled probes shipped within one working day |
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| PrimeTime qPCR Assays are offered in three different sizes to meet any qPCR experimental need. In addition, for the Standard and XL sizes, selection of dye-quencher combination and primer to probe ratio can be specified to meet unique experimental demands. | | Assays consist of a forward primer, a reverse primer, and a dual labeled probe all delivered in a single tube. Each Assay is made to order with estimated shipping in 2 to 4 days from order receipt. Each oligo undergoes 100% QC by mass spectrometry and all QC results are provided free of charge to the customer on the IDT website. | | PrimeTime qPCR Assay Configuration | | Reactions (20µL) | Price
(FAM-Iowa Black FQ) | Price
(other dye-quencher combinations)1 | Estimated Ship Date | Probe (nmoles) | Primers (nmoles)2 |
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| PrimeTime™ Mini qPCR Assay | 100 | $75.00 USD | N/A | 2-4 business days | 0.5 | 1.0 | | PrimeTime™ Standard qPCR Assay | 500 | $120.00 USD | $150.00 USD | 2-4 business days | 2.5 | 2.5-10 | | PrimeTime™ XL qPCR Assay | 2500 | $350.00 USD | $400.00 USD | 2-4 business days | 12.5 | 12.5-50 |
| 1 See table below for available dye quencher combinations
2 The primer to probe ratio may be specified by the customer except for the PrimeTime Mini. | | Available Dye and Quencher Combinations for PrimeTime qPCR Assays | | 5' Dye | 3' Quencher | Mini | Standard | XL |
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| FAM | Iowa Black FQ | • | • | • | | FAM | TAMRA | | • | • | | HEX | Iowa Black FQ | | • | • | | TET | Iowa Black FQ | | • | • | | Cy5 | Iowa Black RQ | | • | • |
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| PrimeTime Dual Labeled Probes are non-extendable oligonucleotides labeled with a 5’ fluorescent reporter and a 3’ quencher dye. The probes are licensed for use in the 5’ Nuclease Real-Time PCR assays. | |
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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. | | |
| 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. | | Product | Qiagen QuantiTect Probe PCR Kit | ABI TaqMan® Gene Expression Master Mix | Bio-Rad iTaq™ Supermix with ROX | Stratagene Brilliant II® QPCR Master mix | Invitrogen Express qPCR SuperMix |
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| 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 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. | | |
| 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 |
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| 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 | | | |  |  |  |  |  | | | | E2F1 | JAK2 | PDK1 | TEC | TNFRSF1A |
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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 |
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| 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 | | | |  |  |  |  |  | | | | TNFRSF1A | PDK1 | JAK2 | E2F1 | TEC |
| | | 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. | |
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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 |
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| 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. | |
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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.
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In step two, the polymerase extends from the primers and begins DNA synthesis.
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| 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. |
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| Setting the primer to probe ratio allows for complete oligo concentration flexibility. Researchers may want specific primer to probe ratios for a number of reasons.: |
Improved Multiplexing
In a multiplex reaction, the master mix reagents can get depleted fairly quickly for an endogenous control, or other highly expressed genes, due to the limited amount of reaction components. Therefore, for these assays in multiplex, it is preferable to limit the primer concentration. If the primers are limited, the reaction will be stopped soon after crossing the threshold, which should improve performance of the lower expressing targets.
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Known Assay Cross Reactivity
In some, rare circumstances with difficult designs or nontraditional applications, cross reactivity may be observed. If this is the case, primers can inadvertently limit the reaction. Sometimes increasing the primer to probe ratio to three or four may help reduce the impact on assay performance.
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Data Recreation
If an assay has been validated under a specific primer to probe ratio, it may be preferable to continue using the same ratio.
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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.
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| 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
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Quenchers
Traditionally, TAMRA has been used as a quencher when the reporter dye is FAM. However, second generation dark quenchers have been developed to reduce background fluorescence. Probes incorporating dark quenchers have lower background fluorescence and can therefore provide greater sensitivity. Dark quenchers absorb broadly and do not emit light allowing for use of multiple reporter dyes with a given quencher. This characteristic allows expanded options for multiplex assays. Dark quenchers simplify detection which makes them compatible with a broad range of image analysis instruments. In addition to supplying commonly used dark quenchers, IDT offers two proprietary dark quenchers, Iowa Black FQ and Iowa Black RQ. Please see Quencher Compatibility Table
below for dye compatibility with dark quenchers.
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Related Information
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