Related DECODED Articles

Do Your qPCR Assays Come With Sequence Information? They Should. Here Is Why.

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:

  1. 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.
    • Helps identify and avoid underlying SNPs—PrimeTime Predesigned qPCR Assay designs are updated with the latest NCBI build to account for the rapid increase in SNP identification [1]. Our assays are synthesized only upon receipt of order, so assays from IDT are designed to avoid both prviously identified and newly discovered SNPs. Stocked qPCR assays purchased from other companies are likely designed based on outdated database information, and are typically provided without sequence information. Without primer and probe sequences, you cannot determine whether more recently identified SNPs might explain your qPCR results.
    • 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 [2], and the article, Interpreting Melt Curves: An Indicator, Not a Diagnosis [3].) With sequence information, quick and accurate data interpretation is possible.
    • Allows validation of new transcriptsAs new transcript variants are reported, knowing where your primers are located allows you to determine whether your primers will amplify the new transcript variants.
  2. Gives your publications credibility—Knowing your primer and probe sequences allows you to publish according to the MIQE guidelines [4]. 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” [5].
  3. 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

  1. Prediger E. IDT. (2013) When Designing PCR Assays, Always Check For SNPs. http://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2013/07/05/when-designing-pcr-assays-always-check-for-snps.
  2. Brookman-Amissah N, Packer H, Prediger E, Sabel J. IDT. (2012) qPCR Assay Application Guide. http://www.idtdna.com/pages/landing/qpcr/guide.
  3. 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.
  4. 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.

  5. Bustin SA. (2012) MIQE Guidelines: A Roadmap for Proper qPCR Experimental Design and Reporting. www.idtdna.com. MP4 Video.

Further Reading
Browse the Table of Contents of our DECODED 4.2—Special qPCR Issuea collection of articles addressing experimental setup; primer and probe design and handling; data analysis; and special applications, such as multiplex and digital PCR.