Of course you need to know the sequences of your qPCR primers and probe—and IDT has always provided them. When you purchase an PrimeTime® Predesigned qPCR Assay from IDT, we disclose primer and probe sequences. Other suppliers often only provide a general chromosomal location without disclosing sequence information. Here is why we think having sequence information is so important:
- Allows you to fully plan and analyze your experiments
- Provides confidence in sequence specificity—IDT® PrimeTime Predesigned qPCR Assays are designed to avoid nonspecific amplification and have undergone a thorough BLAST search against current NCBI databases. However, with the large volume of new sequence data being generated, these databases are frequently updated with additional information. By providing primer and probe sequences, we enable you to interrogate the databases in the future and understand your data in the context of new transcript information.
- Facilitates design of multiplex experiments—When multiplexing with several primer and probe sets in a single assay, it is critical to know your primer and probe sequences to avoid primer-primer and primer-probe interactions.
- Assists data interpretation—Troubleshooting unexpected data can be difficult and time-consuming, and these efforts are exacerbated when you lack sequence knowledge. Identifying why an experiment fails often requires knowledge of sequence properties—the Tm of an oligo, potential dimer or secondary structure formation, or nonspecific amplification. (For qPCR assay troubleshooting support, see Chapter 7 of the IDT PrimeTime® qPCR Assay Application Guide , and the article, Interpreting melt curves: An indicator, not a diagnosis .) With sequence information, quick and accurate data interpretation is possible.
- Allows validation of new transcripts—As new transcript variants are reported, knowing where your primers are located allows you to determine whether your primers will amplify the new transcript variants.
- Gives your publications credibility—Knowing your primer and probe sequences allows you to publish according to the MIQE guidelines . The MIQE guidelines call for providing sequence information in publications for transparency, so that others can better evaluate, repeat, and validate your work. Dr Stephen Bustin, one of the authors of the MIQE guidelines, notes, “Incomplete reporting of experimental detail confounds assessment of qPCR data validity, calling into question scientific conclusions that serve as a basis for further basic research and diagnostic applications” .
- Provides the value you paid for—The sequences of your primers and probe should not be withheld from you. The worth of an assay goes beyond oligo quality and should include the value of sequence identity.
When you are evaluating qPCR assays for purchase, be sure to ask whether you are getting assays designed against the most up-to-date sequence information, and whether you will be given the sequences for your primers and probe. With IDT qPCR assays, you will get both.
References and additional resources
- Brookman-Amissah N, Packer H, Prediger E, Sabel J. IDT. (2012) qPCR Assay Application Guide. http://www.idtdna.com/pages/landing/qpcr/guide.
- Downey N. (2014) Interpreting melt curves: An indicator, not a diagnosis. https://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2014/01/20/interpreting-melt-curves-an-indicator-not-a-diagnosis.
- MIQE Guidelines:
Bustin SA, Benes V, et al. (2009) The MIQE Guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem, 55(4):611−622.
Bustin SA, Beaulieu JF, et al. (2010) MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Mol Biol,11:74–78.
Bustin SA, Benes V, et al. (2011) Primer sequence disclosure: A clarification of the MIQE Guidelines. Clin Chem, 57:919−921.
- Bustin SA. (2012) MIQE Guidelines: A Roadmap for Proper qPCR Experimental Design and Reporting. www.idtdna.com. MP4 Video.
Author: Ellen Prediger, PhD, is a senior scientific writer at IDT.
Product focus: qPCR Reagents—everything but your sample
All the reagents you need for successful qPCR assays are available through IDT.
- Master mix
- Both probe-based and primer only qPCR assays
- Water and buffer
Explore IDT free, online tools for qPCR probe design and analysis. The design engines for these tools use sophisticated formulas that, for example, take into account nearest neighbor analysis to calculate Tm, and provide the very best qPCR assay designs.
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Decrease qPCR background, improve qPCR signal—Review this data demonstrating that probes containing a second, internal quencher provide increased signal detection and greater assay sensitivity in qPCR assays vs. single-quenched probes, such as probes with BHQ Quenchers. ZEN™ and TAO™ Double-Quenched Probes also make it more feasible to use longer probes due to the resulting lower background fluorescence. See the data yourself!
Choosing a qPCR assay: Inventoried or predesigned?—You may know that several companies offer inventoried qPCR assays. But did you know that these assays are often pre-manufactured and stocked, waiting perhaps for years for that order to be placed? In contrast, IDT PrimeTime® Predesigned qPCR Assays are manufactured at the time of order, and can therefore take into account sequence updates such new annotations.
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Browse the PCR and qPCR section of our DECODED Online newsletter for articles addressing experimental setup; primer and probe design and handling; data analysis; and special applications, such as multiplex and digital PCR.