We’ve updated our DECODED article library!

Get answers to your research questions, with articles sorted by application. Try it now »

Choosing a qPCR assay: Inventoried or predesigned?

Several companies offer inventoried qPCR assays that have not only been predesigned, but are also premanufactured and stocked, waiting for that order to be placed. In contrast, IDT PrimeTime® Predesigned qPCR Assays are not pre-manufactured. The designs and have undergone rigorous bioinformatics assessments using sophisticated design algorithms to generate the most efficient assays for a particular genetic region. 

Stocked assays miss updates

Assays sitting in inventory were not necessarily designed with the latest target sequence information available. Genetic information is continually being updated and revised as new SNPs and other sequence elements are discovered—the number of new human SNPs discovered has doubled just in the past 2 years (taken from NCBI dbSNP). No one wants to use an outdated assay that might fall over a newly discovered SNP. Primers designed on a SNP location can significantly affect primer Tm and, consequently, primer efficiency. For the most accurate qPCR data, researchers must take this updated sequence information into account.

Updated sequence and quick synthesis

The IDT design engine uses the most current, clean, and complete target sequence information, that updates with each RefSeq release, to ensure that researchers always receive assays with the most accurate sequence available. The assays are designed but not stocked, so the designs can be updated as new sequence information becomes available. IDT high-throughput manufacturing capabilities are able to produce these assays on demand with quick turnaround time at a cost even lower than other companies’ inventoried assays. By selecting PrimeTime Predesigned qPCR Assays from IDT, researchers not only receive the best, most up-to-date designs, but also are able to receive their assay orders in 2−4 business days.

Lower Cq, higher ΔRn

In a comparison of PrimeTime Predesigned qPCR assays to current market inventoried and custom assays, 25 IDT PrimeTime Predesigned Assays were run in parallel with equivalent Competitor A qPCR 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 qPCR Assays and Competitor A assays were selected to span the same exon boundary of each gene. Each assay was analyzed over five 4-fold dilutions of sample cDNA. The reactions were run with the Applied Biosystems Gene Expression Master Mix and identical thresholds were set for all runs. Data for matched assays is shown in Figure 1. Click here to see results for all 25 matched assay pairs.

The mean efficiency for all IDT assays was 95.55%, with R2 values >0.99. This average efficiency was not only higher than that for the Competitor A assays, but the distribution of calculated efficiencies was also greatly reduced. PrimeTime Predesigned qPCR Assays averaged 0.90 Cq lower than the matched, inventoried assays from Competitor A and ΔRn values were over 30% higher. The data demonstrate that including the best design possible based on current sequence information along with high-quality manufacturing can increase qPCR sensitivity.

Smart positioning, flexible selection

PrimeTime Predesigned qPCR Assays are designed to target exon boundaries or span exon junctions. The IDT web-based ordering system allows users to filter, sort, and select assays based on characteristics like exon location, splice variant targets, RefSeq number, or gene symbol. PrimeTime Predesigned qPCR Assays are available for the majority of human, mouse, and rat transcripts in the NCBI database—where primer and probe designs meet our strict selection criteria.

What you get

All PrimeTime Predesigned qPCR Assays consist of two primers and a hydrolysis probe and come in three sizes with multiple dye-quencher combinations, primer-to-probe ratios, and qPCR design parameters. Each oligo undergoes 100% QC by mass spectrometry, with QC results provided free of charge on the IDT website. 

Learn more about IDT PrimeTime Predesigned qPCR Assays and custom PrimeTime qPCR Assays.

 PrimeTime Pre-designed Assays Competitor A Assays
 IDT NM_005911
Competitor NM_005911

Figure 1. PrimeTime® Predesigned qPCR Assays are more sensitive than competitor A inventoried assays. 25 randomly selected PrimeTime Predesigned qPCR Assays were compared to equivalent assays from Competitor A. Each assay was analyzed over a 4-fold dilution series (50 ng to 0.195 ng) universal human reference (UHR) cDNA, using Applied Biosystems TaqMan® Gene Expression Master Mix. Reactions were run on the ABI 7900HT Fast Real-Time PCR System 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 3 Competitor A assays and the equivalent IDT PrimeTime Predesigned qPCR Assays is shown. Efficiency measurements were calculated from the standard curve. The average reaction efficiency was not only higher for the IDT PrimeTime Predesigned qPCR Assays compared to the Competitor A assays, but the distribution of calculated efficiencies was also greatly reduced. See the data for all 25 assay pairs.

Related reading

DECODED 4.2—Special qPCR Issue—Get this compendium of IDT articles with qPCR tips, troubleshooting advice, and researcher examples in one convenient volume.
PrimeTime® qPCR Application GuideDownload this useful resource that provides experimental overviews, protocols, data analysis, and troubleshooting chapters.
How to avoid false positives in PCR and what to do if you get them—Learn about common causes of false positives in the Negative Template Control sample during PCR, and suggestions for preventing them.

Starting with RNA—one-step or two-step RT-qPCR?—Trying to decide whether to use a one-step protocol that combines the RT reaction and PCR in one tube, or a two-step protocol where the RT reaction is performed separately from the PCR? Here are some guidelines. 
Multiplex qPCR—how to get started—Learn how multiplex qPCR can save sample, reagent cost, and time. Read these recommendations for multiplex qPCR assay design and experimental setup. 

Author: Ellen Prediger is the Director of Scientific Communication at IDT.

© 2011 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.