qPCR: Guidelines for Validating a Multiplex Assay

Quantifying expression of multiple target genes in the same qPCR reaction well, also known as multiplexing, has many advantages. Multiplexing can save setup time, reduce cost and minimize the amount of sample consumed in each experiment.

However, there are some important considerations and additional work that must be done up front to make sure you get the most out of your multiplex assay, especially when validating one for the first time.

When validating a multiplex assay, you need to make sure that several essential factors of assay performance are unaffected by the presence of other primer/probe sets. Those factors include quantification cycle (Cq) values, linearity, sensitivity and efficiency for each target.

Interference from other amplifications occurring within the reaction well can lead to poor or biased amplification and skew quantification results. This guide will take you step-by-step through the process of validating a qPCR multiplex assay to provide optimal results.


Bustin, S.A. et al. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 55, 1–12. Additional Resource Refer to GoTaq® Probe qPCR Master Mix Technical Manual #TM378 for more details.


Samantha Lewis, PhD, Promega Corporation

About PromegaPromega Corporation

With a portfolio of more than 3,000 products covering the fields of genomics, protein analysis and expression, cellular analysis, drug discovery and genetic identity, Promega is a global leader in providing innovative solutions and technical support to life scientists in academic, industrial and government settings.

Promega products are used by life scientists who are asking fundamental questions about biological processes as well as by scientists who are applying scientific knowledge to diagnose and treat diseases, discover new therapeutics, and use genetics and DNA testing for human identification.

Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and iform site visitors interested in medical research, science, medical devices and treatments.

Last updated: Jun 8, 2023 at 9:03 AM


Please use one of the following formats to cite this article in your essay, paper or report:

  • APA

    Promega Corporation. (2023, June 08). qPCR: Guidelines for Validating a Multiplex Assay. News-Medical. Retrieved on June 14, 2024 from https://www.news-medical.net/whitepaper/20180206/qPCR-Guidelines-for-Validating-a-Multiplex-Assay.aspx.

  • MLA

    Promega Corporation. "qPCR: Guidelines for Validating a Multiplex Assay". News-Medical. 14 June 2024. <https://www.news-medical.net/whitepaper/20180206/qPCR-Guidelines-for-Validating-a-Multiplex-Assay.aspx>.

  • Chicago

    Promega Corporation. "qPCR: Guidelines for Validating a Multiplex Assay". News-Medical. https://www.news-medical.net/whitepaper/20180206/qPCR-Guidelines-for-Validating-a-Multiplex-Assay.aspx. (accessed June 14, 2024).

  • Harvard

    Promega Corporation. 2023. qPCR: Guidelines for Validating a Multiplex Assay. News-Medical, viewed 14 June 2024, https://www.news-medical.net/whitepaper/20180206/qPCR-Guidelines-for-Validating-a-Multiplex-Assay.aspx.

Other White Papers by this Supplier

While we only use edited and approved content for Azthena answers, it may on occasions provide incorrect responses. Please confirm any data provided with the related suppliers or authors. We do not provide medical advice, if you search for medical information you must always consult a medical professional before acting on any information provided.

Your questions, but not your email details will be shared with OpenAI and retained for 30 days in accordance with their privacy principles.

Please do not ask questions that use sensitive or confidential information.

Read the full Terms & Conditions.