PrimeTime Pre-designed qpcr Assays
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1 PrimeTime Pre-designed qpcr Assays nuclease assays for human, mouse, and rat PrimeTime qpcr Assays IDT now offers PrimeTime Pre-designed qpcr Assays that are guaranteed to work for human, mouse, and rat transcriptomes! PrimeTime qpcr Assays consist of two primers and a hydrolysis probe. All three components are combined into a single tube and shipped in business days. Each oligo undergoes 1% QC by mass spectrometry, with all QC results provided free of charge on the IDT website. Performance Guaranteed PrimeTime Pre-designed qpcr Assays are guaranteed to provide >9% efficiency when using Assays generated from the IDT Pre-designed Assay collection, a commercially available master mix, and measured over a minimum of four orders of magnitude. Users routinely experience lower Cq values, higher qpcr efficiency, and larger ΔRn with PrimeTime Pre-designed qpcr Assays than with competitor products. Ultimate Flexibility Select from five different dye/quencher combinations and choose a premixed primer:probe ratio from 1:1 to :1 to maximize experimental flexibility and multiplexing capabilities. Choose from three different scales to best fit your experimental needs and reduce the number of unused reactions. Easy Online Ordering Filter and sort by assay characteristics like exon location, splice variant targets, RefSeq number, or gene symbol. The ordering system contains Assays designed to target exon boundaries for every human, mouse, and rat transcript in the NCBI database.
2 PrimeTime Pre-designed qpcr Assays Design Process IDT s design engine incorporates many parameters to produce the best possible PrimeTime Predesigned qpcr Assay. The design process uses the most current, clean, and complete target sequence information and stresses accurate Tm prediction and protection against off-target amplification. The process includes a wealth of additional secondary design considerations and design scoring criteria focused on isolating the best performance potential for a specific target design. Current target sequence information Access the most current sequence data available The IDT design engine runs against the cleanest possible target data that include up-to-date SNP and intron/exon junction locations. The design libraries are continually refreshed as new sequence data become available. Melting temperature (Tm) Design assays with accurate Tm for primers and probes Accurate prediction of Tm is essential for precise PCR performance. The assay design engine incorporates the most currently available Tm prediction algorithms and nearest-neighbor parameter sets. IDT continues innovative DNA thermodynamics research and integrates those improvements into its calculation and oligonucleotide design tools. Cross reaction and off-target amplification prevention Prevent cross reaction and off-target amplification, mask SNPs Proprietary assay design engine algorithms and BLAST alignments check the target sequence to protect against cross reaction and off-target amplification. The algorithms reduce potential off-target amplicon formation, as well as individual element non-specificity that can compromise efficiency by consuming assay components through off-target interactions. The Assay design engine ensures that primers or probes are not placed on SNP locations. Score remaining assays and publish to web Scoring and Publishing The main assay design engine algorithm is tuned to aggressively over-design. This produces dozens of possible assay options for each target which are then carefully weighed for selection of those with the best overall performance and reliability characteristics. These selection criteria include examination of characteristics such as poly runs, short sequence repeats, and hetero-dimer and homo-dimer folding energies.
3 Validation of the Design Engine To validate the design parameters, IDT tested 1 randomly selected PrimeTime Pre-designed qpcr Assays. Each assay was analyzed with a log dilution series from ng to pg UHR cdna. Efficiency measurements were calculated from the standard curve. All experiments were performed on the ABI 79HT Fast Real-Time PCR System with the Applied Biosystems Gene Expression Master Mix. Result Highlights More than 99% of the Assays tested exhibited >9% efficiency. 1% of the Assays had R values >.99. The mean efficiency of all Assays was 9.%. Over /3 of all Assays had >9% efficiency. 3% Distribution of Assay Efficiency % Figure 1. IDT Assay Selection Engine Generates Highly Efficient qpcr Assays. 1 Assays were selected randomly and analyzed with a -fold standard curve of UHR cdna. Each reaction was tested with nm of each primer and nm FAM/ZEN/IBFQ probe with the Applied Biosystems TaqMan Gene Expression Master Mix. The reactions were run on the ABI 79HT Fast Real-Time PCR System with the following PCR cycling conditions: min. C; 1 min. 9 C; x (1 sec. 9 C, 1 min. C). Frequency % 1% 1% % % Calculated Efficiency (%) Pre-designed Assays Work With All Master Mixes Tested 1 different PrimeTime Pre-designed qpcr Assays were tested with commercially available master mixes. A minigene plasmid was used as a template and diluted over logs to calculate efficiency. The reactions were all run on the ABI 79HT instrument at the recommended concentration and cycling conditions for each master mix. All Assays exhibited greater than 97% efficiency with a 1% success rate. Commercial Master Mix Mean Efficiency (%) Percent Success Agilent Brilliant III Ultra Fast QPCR Applied Biosystems TaqMan Gene Expression Invitrogen SuperMix Qiagen QuantiTect Probe PCR Kit BioRad itaq Supermix with ROX 1. 1 BioSciences PerfeCTa qpcr 97. 1
4 PrimeTime Pre-designed Assays Outperform Competitor Products Performance Highlights Improved Results for Fast Cycling With PrimeTime Pre-designed qpcr Assays and Agilent s Brilliant III Master Mix, you no longer have to sacrifice performance for speed. Increased Sensitivity On average, PrimeTime Pre-designed qpcr Assays were nearly one full Cq lower and had endpoint signals more than 3% greater than the competition. (Figures and 3). Higher qpcr Efficiency When compared to, not only did PrimeTime Pre-designed Assays have a higher average efficiency but the distribution of calculated efficiencies was also greatly reduced. (Figure ). PrimeTime Pre-designed 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 Pre-designed qpcr Assays were tested using the Agilent Brilliant III Ultra Fast QPCR Master Mix, which allows run times as short as minutes. These results were compared to matched, inventoried assays from. No sacrifice in efficiency. All maintained efficiencies between 9 1%. of the assays had efficiency values <9%. Greater sensitivity. PrimeTime Pre-designed qpcr Assays had lower Cq values compared to matched, inventoried assays from by over. on average and ΔRn values that were almost % higher. WDR3 (NM_7) TIMP3 (NM_3) HERC1 (NM_39) Baselined Signal (delta Rn) 1 Baselined Signal (delta Rn) 7 3 Baselined Signal (delta Rn) WDR3 TIMP3 HERC1 Mean Cq ( ng) Efficiency Endpoint (ΔRn) PrimeTime PrimeTime 97.% 9.% 9.9% 9.% 91.7%.% PrimeTime Figure. PrimeTime Pre-designed qpcr Assays Maintain Efficiency Under Fast-Cycling Conditions. PrimeTime Pre-designed qpcr Assays were compared to equivalent assays designed to span the same exon boundaries. The reactions used -fold dilutions of cdna template (minigenes, IDT) and Brilliant III Ultra Fast QPCR Master Mix (Agilent) and were run on the ABI 79HT Fast Real-Time PCR System with the following PCR cycling conditions: 3 min. 9 C; x ( sec. 9 C, 1 sec. C). Identical thresholds were set for comparison across assays. 3 matched assays are shown: WDR3 (NM_7), TIMP3 (NM_3), and HERC1 (NM_39). For each of the parameters shown (mean C q, efficiency, and endpoint data), the PrimeTime Pre-designed qpcr Assays outperformed the assays.
5 Increased Sensitivity assays from were compared to an equal number of IDT s PrimeTime Pre-designed qpcr Assays. The assays consisted of 1 inventoried pre-designed assays and 1 made-to-order pre-designed assays. To ensure an accurate comparison was made, the and assays were selected to span the same exon boundary of each gene. The reactions were run with the Applied Biosystems Gene Expression Master Mix and identical thresholds were set for all runs (Figures 3 and ). Rn (FAM/ROX) 3 1 Comparison of Assay Performance with WDR3 (NM_7) Figure 3. Are More Sensitive than Assays. PrimeTime Pre-designed qpcr Assays were compared to equivalent assays using -fold dilutions of cdna template and the Applied Biosystems TaqMan Gene Expression Master Mix. The reactions were run on the ABI 79HT Fast Real- Time PCR System with the following PCR cycling conditions: min. C; 1 min. 9 C; x (1 sec. 9 C, 1 min. C). Identical thresholds were set for all runs for comparison across assays. A comparison of the WDR3 (NM_7) assay and the equivalent IDT PrimeTime Pre-designed qpcr Assay is shown Mean Cq Value NM_3 NM_31 NM_1 NM_39 NM_3 NM_33 NM_1 NM_911 NM_3 NM_7 Comparison of Mean Cq Value NM_11 NM_137 NM_7 NM_ NM_71 NM_39 NM_3 Gene RefSeq Accession Number NM_7 NM_11 NM_7 NM_1 NM_13 NM_17 NM_1 NM_119 PrimeTime Figure. PrimeTime Pre-designed qpcr Assays Have Consistently Lower C q Values for the Same Target. PrimeTime Pre-designed qpcr Assays were compared to equivalent Competitor A assays using -fold dilutions of UHR cdna and the Applied Biosystems TaqMan Gene Expression Master Mix. The reactions were run on the ABI 79HT Fast Real-Time PCR System with the following PCR cycling conditions: min. C; 1 min. 9 C; x (1 sec. 9 C, 1 min. C). Identical thresholds were set for all runs for comparison across assays. The mean C q values from the ng dilution of UHR cdna are shown. *Successful assays were considered to be those that demonstrated >9% efficiency when measured over logs with R values >.99. Higher qpcr Efficiency qpcr efficiency was measured using a comparison of and IDT assays for sensitivity (Figure ). Again, the PrimeTime Pre-designed qpcr Assays have a higher average qpcr efficiency than assays. In addition, the overall distribution of qpcr efficiency was narrower and higher than that for assays. Figure. PrimeTime Pre-designed qpcr Assays Have Higher qpcr Efficiency and a Smaller Distribution Range than Assays. PrimeTime qpcr Assays were compared to matched assays using -fold dilutions of cdna and the Applied Biosystems TaqMan Gene Expression Master Mix. The reactions were run on the ABI 79HT Fast Real- Time PCR System with the following PCR cycling conditions: min. C; 1 min. 9 C; x (1 sec. 9 C, 1 min. C). Identical thresholds were set for all runs for comparison across assays. Frequency Comparison of qpcr Efficiency PrimeTime qpcr Efficiency (%)
6 Ordering PrimeTime Pre-designed Assays 1. Enter the gene name, RefSeq number, or Assay ID.. Select the species from mouse, human, or rat. 3. Click on Submit.. To sort the results, drag the column that you would like to sort by to the top. Drag more than one column to the top to further refine the search.. Select the Assay you would like to order.. Click on Customize to select the size (Mini, Standard, or XL), the dye-quencher combination, and the primer-to-probe ratio. 7. Click Add To Order or Add And Checkout button. A B D C Enhanced Selection Features A. Group by symbol, species, or splice variant by dragging the column title to the indicated space. B. Filter by species, gene symbol, or splice variant. C. Sort results by recommended Assays. When this is selected, the first Assay in the list will be the highest ranked and most recommended based on IDT scoring parameters. D. View RefSeq numbers detected for each Assay design PrimeTime products are licensed by Applied Biosystems for use in the Nuclease Assay for non-commercial research purposes only. A license to perform the patented Nuclease Process for research is obtained by the purchase of (i) both Licensed Probe and Authorized Nuclease Core Kit, (ii) a Licensed Nuclease Kit, or (iii) license rights from Applied Biosystems. This product contains Licensed Probe. Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US:,3,,,73,91,,7,93,,3,77,,,9, and,,37 (claims 1-1 only). The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research. The right to use this product in the Nuclease Process under the applicable claims of US Patents Nos.,1,1 and,7,97, and corresponding patent claims outside the United States, can be obtained through purchase of an Authorized Nuclease Core Kit. Certain PrimeTime Assays may contain one or more proprietary dyes that are sold subject to separate limited licenses. Cy, is a registered trademark of GE HealthCare, and when applicable, the purchaser obtains a limited non-exclusive sublicense under U.S Patent Nos. 99 and and foreign equivalent patents and other foreign and U.S counterpart applications to use the Product to perform research. NO OTHER LICENSE IS GRANTED EXPRESSLY, IMPLIEDLY OR BY ESTOPPEL. Use of the Product for commercial purposes is strictly prohibited without written permission from GE HealthCare. Iowa Black is a registered trademark of IDT. The purchase of this product includes a limited non-exclusive sublicense under U.S. Patent Nos , and 7 3 and U.S counterpart applications for use in the purchaser s internal research applications only. For more information go to 11 Integrated DNA Technologies
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