Common TaqMan Myths Busted
Fact #1: TaqMan Assays can be used on any qPCR instrument.
Quantitative PCR can refer to a real-time or digital PCR experiment. Many different models of qPCR instruments are available. Each model must have an excitation source to excite the fluorescent dye and a detector to detect the fluorescent emissions. The excitation and emission filters support different types of dyes, and the number of these filters can vary depending upon the qPCR instrument model chosen. In addition, each model must have a thermal block, which can be either fixed or interchangeable. With digital PCR, the thermal cyclers are separate from the reader/imager component in most cases.
With tens of thousands of peer-reviewed publications, the 5’ nuclease assay using TaqMan probes is one of the most widely used detection methods for real-time or digital PCR. Many of the publications demonstrate that TaqMan Assays and reagents can be used on instruments other than Applied Biosystems qPCR instruments, including Bio-Rad, Roche, and Agilent qPCR systems.
Fact #2: High specificity is the hallmark trait of TaqMan Assays.
The mechanisms of SYBR™ and TaqMan detection are inherently different. SYBR stain is an intercalating dye that binds to double-stranded DNA (dsDNA) and is used to detect PCR product as it accumulates during PCR. To distinguish specific from nonspecific product, a melt curve analysis is required post-PCR.
On the other hand, TaqMan Assays utilize sequence-specific, fluorogenic-labeled probes to detect a target as it accumulates during PCR. The TaqMan detection method drives higher specificity due to the sequence specificity of the TaqMan probe and the three binding events in close proximity required to generate the signal.
Fact #3: TaqMan Assays are simple to order and use, and can be used by any scientist—no matter their level of experience.
With over 10 million predesigned assays to choose from, TaqMan Assays have a comprehensive set of products available for gene expression, noncoding RNA, copy number variation, SNP genotyping, mutation detection, and protein expression analysis. Predesigned TaqMan Assays do not require optimization or verification, are guaranteed to work,* and are suitable for all users. Just add your sample to the predesigned TaqMan Assay and master mix, and you are ready to go.
In fact, it has never been easier to search and purchase predesigned TaqMan Assays through our website. Visit the Real-Time PCR Assays order page at thermofisher.com/ordertaqman. Simply choose your species, type in your gene of interest, and select your assay. If you want to learn more about a specific assay or understand which assay gives you the best coverage, you can find this information through a simple click of your mouse.
Fact #4: We guarantee* the performance of ALL predesigned TaqMan Assays.
We have proven expertise and years of assay design and manufacturing experience. Every assay is designed using our sophisticated bioinformatics design pipeline that was initially verified by lab-testing over 18,000 assays, and has been further verified by thousands of peer-reviewed publications.
Our gene expression, noncoding RNA, SNP genotyping, copy number, drug metabolism enzyme, mutation detection, and protein assays enable you to obtain high quality and performance. If a TaqMan Assay does not perform according to the specifications of the guarantee, we’ll replace it at no cost, or we’ll credit your account*. Find out more at thermofisher.com/taqmanguarantee.
Did you know?
PCR efficiency is a critical factor in real-time PCR assay design and optimization. A standard curve (generated from a dilution series of the target template) is used to obtain an efficiency value. This efficiency value acts as a marker of overall reaction “health.” It is fairly simple to determine the efficiency by calculating the slope of a standard curve. Alternatively, the software on our newer Applied Biosystems™ Real-Time PCR systems can calculate the efficiency for you. However, if there are mistakes in preparing the dilution series, this could impact the accuracy of your PCR efficiency.
Fact #5: Applied Biosystems is your trusted brand for real-time PCR, and we’ve made it simple to understand TaqMan methodology.
The Applied Biosystems™ brand is associated with over 20 years of experience in real-time PCR, with TaqMan Assays leading the way in gold-standard performance, quality, and content. Developed using longstanding bioinformatics expertise in primer and probe design and stringent testing across applications and integrated platforms, TaqMan Assays provide you with reliable and robust real-time PCR solutions. We realize that getting started with TaqMan Assays may be daunting, so we have made it simple by providing tools for education, selection, and technical support to guide you along every step of the way.
Fact #6: Time is money, too… predesigned TaqMan Assays are optimized to work every time.
The amount of time you’ll save using TaqMan Assays actually provides a big savings when you consider the time you’re spending verifying assays and the reagent costs to do that. Remember that time is money, and verifying SYBR Green assays (i.e., design, test, redesign, etc.) or assays from inexperienced providers requires significant time and reagents. We stand behind every TaqMan Assay you buy.
TaqMan Assays are covered in the TaqMan Assays qPCR Guarantee program. If you do encounter a problem, simply report it to our technical support team, who will help you identify the issue and replace the assays as needed. Visit thermofisher.com/taqmanguarantee for full details.
Fact #7: TaqMan Assays comply with the recommendations proposed by the MIQE authors.
MIQE, or the Minimum Information for Publication of Quantitative Real-Time PCR Experiments, is a set of guidelines originally published by Bustin et al. in Clinical Chemistry (April 2009) to “describe the minimum information necessary for evaluating qPCR experiments.” These recommendations cover all aspects of qPCR experimental design and analysis, as well as standards for sharing information with colleagues to ensure transparency and reproducibility.
The original guidelines recommended that the primer sequences needed to be included in all real-time PCR publications. However, many commercial qPCR assays do not include the primer/probe sequences due to this being commercially sensitive information. An amendment to the article was published in 2011 to clarify the stipulation of primer sequence disclosure, stating “EITHER primer sequences OR amplicon context sequence” should be included.
All predesigned TaqMan Assays are designed using an advanced and proprietary bioinformatics pipeline that helps ensure target specificity, maximum amplification efficiency, and standardized running conditions requiring no optimization. In compliance with current MIQE guidelines, TaqMan Assays are provided with the context sequence, the probe location, and—if appropriate—the exon-exon boundary that the assay crosses. Every TaqMan Assay design has a unique assay ID number that can be used as a reference with colleagues, or in publications to replicate the design.
*Terms and conditions apply. For complete details, go to thermofisher.com/taqmanguarantee
For Research Use Only. Not for use in diagnostic procedures.