qPCR: Guidelines for Validating a Multiplex Assay

Introduction

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.

Reference

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.

Acknowledgements

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.


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Last updated: Jul 14, 2018 at 7:08 PM

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