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Documentation and Support qPCR Application Guide PrimeTime qPCR Assay Product Brochure ZEN Double-Quenched Probe Overview Frequently Asked Questions PrimeTime qPCR Assay Resuspension Protocol

NEW! IDT and Agilent are working together to improve your qPCR research. By working with Agilent Mx QPCR systems, Brilliant qPCR reagents, and IDT PrimeTime Assays, you will benefit from utilizing a fully integrated and validated system optimized to function at peak performance. Click here for more information.     NEW! Improve qPCR assay performance with Double-Quenched Probes. Reduce background noise, decrease Cq values and improve precision. To learn more, download the ZEN Double-Quenched Probe Overview.     IDT offers PrimeTime qPCR Assays and qPCR probes designed for 5’ Nuclease assays, the gold standard for quantitative gene expression studies.     PrimeTime qPCR Assays consist of a forward primer, a reverse primer, and a qPCR probe all delivered in a single tube. With the added capability of selecting from multiple dye-quencher combinations, primer to probe ratio, and qPCR design parameters, optimizing qPCR reactions is no longer complicated or expensive.     PrimeTime qPCR Probes are also available individually with a large selection of dye-quencher combinations.

OrderingProductsSupportPerformance Order Using IDT's qPCR Assay Design Tools Order by Entering Design Sequences RealTime PCR Assay Design Tool Validated assay design tool for target sequences contained within the RefSeq database. Guaranteed to provide qPCR efficiency >90% when run under default settings.   PrimerQuest Custom Design Tool Custom primer and probe design tool. Assays for target sequences that are not part of the RefSeq database can be entered. PrimeTime qPCR Assays Primers and probes mixed together and shipped in a single tube. ZEN-Double Quenched Probes now available in assays!   PrimeTime qPCR Probes Select from a wide variety of dyes, quenchers and scales. ZEN-Double Quenched Probes now available!   Express qPCR Probes Express probes shipped within one working day. ZEN-Double Quenched Probes now available! PrimeTime qPCR Assays 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 qPCR 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 PrimeTime™ Mini qPCR Assay 100 $75.00 USD $75.00 USD 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 FAM ZEN/Iowa Black FQ* • • • FAM TAMRA   • • HEX Iowa Black FQ   • • TET Iowa Black FQ   • • Cy5 Iowa Black RQ   • • *ZEN/Iowa Black FQ is a double-quenched probe which provides superior performance to traditional dual-labeled probes. For more information download the ZEN Double-Quenched Probe Overview.   PrimeTime qPCR Probes PrimeTime qPCR DNA probes are non-extendable oligonucleotides labeled with a 5’ fluorescent reporter and a 3’ quencher dye. For more information on dyes and quenchers, please visit the support tab. The probes are licensed for use in the 5’ Nuclease Real-Time PCR assay (with companion dye and quencher licenses). PrimeTime qPCR probes are available with a variety of reporter/quencher combinations and synthesis scales. The prices and estimated turnaround time for these probes will depend on the degree of complexity. Probes at the Standard Level will ship in approximately 3-5 business days while probes at the Complex Level will ship in approximately 4-6 business days. The minimum guarantees are listed for probes that are 15-35 bases in length. Please inquire about yield and price for probes 36-45 bases in length If you require qPCR probes for an application other than the 5’ Nuclease Real-Time PCR assay, please use the DLP builder. PrimeTime qPCR Probes - Standard Level 5' Reporter Dye(s) Quencher Family Synthesis Scale / Minimum Guarantee 100 nmole / 10 nmoles 250 nmole / 25 nmoles 1 umole / 50 nmoles FAM ZEN / Iowa Black FQ * $195.00 USD $275.00 USD $415.00 USD Iowa Black $170.00 USD $240.00 USD $360.00 USD TAMRA $195.00 USD $275.00 USD $415.00 USD Black Hole Quencher $195.00 USD $275.00 USD $415.00 USD HEX TET Iowa Black $210.00 USD $300.00 USD $450.00 USD Black Hole Quencher $245.00 USD $350.00 USD $520.00 USD *ZEN/Iowa Black FQ is a double-quenched probe which provides superior performance to traditional dual-labeled probes. For more information download the ZEN Double-Quenched Probe Overview. The estimated turnaround time for Standard Level Probes is 3-5 business days. PrimeTime qPCR Probes - Complex Level 5' Reporter Dye(s) Quencher Family Synthesis Scale / Minimum Guarantee 100 nmole / 2 nmoles 250 nmole / 8 nmoles 1 umole / 20 nmoles FAM TAMRA NHS Ester N/A $415.00 USD $580.00 USD MAX NHS Cy3 TEX 615 Cy5 TYE 563 TYE 665 Iowa Black $225.00 USD $260.00 USD $475.00 USD Black Hole Quencher $315.00 USD $365.00 USD $545.00 USD JOE NHS Ester TAMRA NHS Ester ROX NHS Ester Texas Red-X NHS Ester Iowa Black N/A $345.00 USD $515.00 USD Black Hole Quencher N/A $420.00 USD $630.00 USD The estimated turnaround time for Complex Level Probes is 4-6 business days. Mini qPCR Probes The PrimeTime Mini qPCR probes are probes available on a small scale. The smaller scale and lower price makes them ideal for trying out a new probe, for screening the expression levels of many genes, or for testing the conversion to FAM/IBFQ and FAM/ZEN/IBFQ from other FAM-related quenchers. Mini probes are offered with a 5’ FAM and the option of a 3’ IBFQ quencher alone or in combination with the internal ZEN quencher. The estimated turnaround time is 3-5 days. PrimeTime Mini qPCR Probes 5' Reporter Dye(s) Quencher(s) Delivery Amount 0.5 nmoles FAM ZEN / Iowa Black FQ * $60.00 USD Iowa Black $60.00 USD *ZEN/Iowa Black FQ is a double-quenched probe which provides superior performance to traditional dual-labeled probes. For more information download the ZEN Double-Quenched Probe Overview. The turnaround time from order to ship for PrimeTime Mini qPCR Probes is 3-5 business days. Express qPCR Probes Express qPCR probes allow researchers the opportunity to have their qPCR probes ship in just one working day! All oligos are HPLC purified and quality control checked by mass spectrometry and the trace is available free on our website. Due to the fast turnaround time, these probes are limited to select dye-quencher combinations and synthesis scales. All Express orders must be placed online by 2:00pm ET. The orders will be shipped with next day priority for delivery by 10:30am. All Express probes must be 18-35 bases, will be HPLC purified and QC verified by mass spectrometry. Probes will be shipped lyophilized and will have a minimum guarantee of 5 nmoles. Additional shipping and handling fees for expedited service apply. PrimeTime Express qPCR Probes 5' Reporter Dye(s) Quencher(s) Synthesis Scale / Minimum Guarantee 100nm / 5 nmoles FAM ZEN / Iowa Black FQ * $249.00 USD Iowa Black $229.00 USD Black Hole Quencher-1 $249.00 USD *ZEN/Iowa Black FQ is a double-quenched probe which provides superior performance to traditional dual-labeled probes. For more information download the ZEN Double-Quenched Probe Overview. The turnaround time from order to ship for Express Dual Labeled Probes is 1 business day. Technical Information Overview of 5’ Nuclease Assays Selecting the Correct qPCR Reporter Dyes and Quenchers ZEN Double-Quenched Probes Selecting the Correct Primer to Probe Ratio Selecting the Correct Master Mix Frequently Asked Questions   Product Documentation PrimeTime qPCR Assay Product Brochure PrimeTime qPCR Assay Resuspension Protocol ZEN Double-Quenched Probe Overview PrimeTime qPCR Assay Performance Dynamic Range and Sensitivity 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.   PrimeTime Reliability with Commercially Available Master Mixes 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 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.   Lot to Lot Assay Reproducibility 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 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 Reproducibility Across Assay Scales 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 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.   Dye-Quencher Combinations 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 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.

Overview of 5' Nuclease Assays 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.   In step two, the polymerase extends from the primers and begins DNA synthesis. 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.

Selecting the appropriate primer to probe ratio. 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 of the higher expressing genes. If the primers are limited, the amplification of the higher expressing genes will decrease, thereby making the mastermix ingredients more available for the amplification of the lower expressing targets. The primer to probe ratios that work best will depend on your particular experiment, but a good starting point is to limit the ratio for the highest expressing genes to 1 and increase the ratio for the lowest expressing genes to 4. Limit 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. 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.

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, 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 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. IDT has developed an internal ZEN quencher that enables to 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 Cq values, improved precision, and enable the use of longer probes for design in AT-rich regions. For more information download the ZEN Double-Quenched Probe Overview. IDT supplies commonly used dark quenchers as well as the proprietary dark quenchers, Iowa Black FQ, Iowa Black RQ, and the newly developed internal ZEN quencher. TAMRA is also a quencher option for a FAM reporter dye. Related Information Instrument Compatibility Table Quencher Compatibility Table Dye and Quencher Wavelength Figure

Double-quenched Probes. IDT has developed a new internal ZEN quencher which enables the production of double-quenched probes with less background and increased signal! Double-quenched probes contain a 5’ FAM fluorophore, a 3’ IBFQ quencher, and an internal ZEN quencher. While traditional probes have around 20-30 bases between the fluorophore and the quencher, the internal ZEN quencher decreases that length to only 9 bases. This shortened distance, particularly when combined with the traditional 3’ end quencher, leads to a much more thorough quenching with much less background and enables the use of much longer probes for designing in AT-rich target regions. In addition to the significantly decreased background, the double-quenched probes also have consistently reduced Cq values and improved precision when compared to traditional probes. Use of the double-quenched probes can allow users to experience both increased sensitivity and precision in their qPCR experiments. For more information download the ZEN Double-Quenched Probe Overview.

IDT recommends the Brilliant II qPCR Master Mix from Agilent. The combination of the Mx3005P qPCR instrument and Brilliant II/III qPCR Master Mix from Agilent and the PrimeTime qPCR Assays from IDT provides a fully supported solution, ideal for qPCR validation studies. This end-to-end qPCR solution has been extensively tested against competitor reagents and assays. Agilent’s Brilliant II qPCR and QRT-PCR Master Mix Kits offer superior sensitivity for improved accuracy and reproducibility of nucleic acid quantification. Combined with the Primetime qPCR Assays, this product set provides the complete solution for gene expression studies and wide array of growing qPCR applications at an affordable price point. For more information on Agilent’s Master Mix kits, please see the Brilliant II or Brilliant III qPCR and qRT-PCR pages. IDT has tested other master mixes and found that PrimeTime qPCR Assays demonstrated efficiency close to 100% across many commercially available master mixes. For more information on master mixes tested, please see the PrimeTime qPCR Assays Technical Bulletin.