Multiplex qPCR—how to get started

Advantages of multiplex qPCR

In multiplex qPCR, multiple targets are amplified in a single reaction tube. Each target is amplified by a different set of primers, and a uniquely-labeled probe distinguishes each PCR amplicon. Thus, you can measure the expression levels of several targets or genes of interest quickly.

To determine whether multiplexing is appropriate for your experiments, consider sample size, reagent cost, and time spent. Multiplex qPCR minimizes the amount of starting material required, which can be of critical value when samples are limited and/or precious. Even when starting material is plentiful, multiplexing can save time by increasing throughput and decreasing sample handling. It can also save on the cost of reagents and other consumables.

Steps for multiplex qPCR assay design

As multiple primers and probes must be designed for this application, we strongly recommend use of a design tool with a validated algorithm. Suitable tools can be found in the SciTools™ applications section of the IDT website. For best results:

Step 1. Select primer and probe sequences. If you are working with human, mouse, or rat targets, IDT has done the work for you by providing a library of predesigned assays (primer and probe sequences) for virtually every sequence in these genomes. This powerful design engine identifies assays using up-to-date sequence information. These assays have been individually BLAST searched across the transcriptome thereby reducing cross reactivity. Select a few assays for each gene of interest for these species, using the PrimeTime™ Predesigned qPCR Assay Selection Tool. If you are working with a species other than human, mouse, or rat, or want to force certain custom design criteria, use the PrimerQuest™ Tool.

Step 2. Analyze primer and probe sequences. Use the OligoAnalyzer™ Tool to identify primer/probe hairpins, homo- or heterodimers, or any primer/probe complementarity across the different assays to be used in the same multiplex qPCR experiment. Avoid extensive 3’ overlap (greater than 4 bases) between primers, otherwise the primers can be consumed in nonproductive amplification. Often, shifting the primers over a few bases will reduce the overlap.

Step 3. Always test assay efficiency. Run each assay in singleplex reactions before conducting multiplex qPCR. This is important to determine that the C_q values obtained are real and not artificially reduced by the multiplex reaction.

Figure 2. Validating Multiplex Reactions. In this assay validation experiment, four 10 μL c-src tyrosine (CSK) qPCR assays were run in triplicate on a CFX384 instrument (Bio-Rad) using 1X PerfeCTa Multiplex qPCR SuperMix (Quanta Biosciences); 500 nM each primer; 250 nM each FAM-labeled probe; and 20, 2, 0.2, and 0.02 ng cDNA made from Universal Human Reference RNA (Agilent). Reactions were performed at 2 min 95°C; 40 x (15 sec 95°C, 45 sec 60°C). Note that for a given assay, C_q values remain virtually unchanged in singleplex and multiplex reactions. The lower fluorescence intensity seen for the multiplex reactions is expected, as reagents are consumed more quickly.

Considerations for multiplex qPCR setup

The experimental design for multiplexing is more complicated than for single-reaction qPCR. Amplification of the multiple targets in a single sample can be influenced by factors including gene expression levels, primer interactions, and competition for reaction reagents. Therefore, careful primer and probe design and optimization of amplification are critical.

1. Check for primer and probe sequence interactions. Use the free IDT OligoAnalyzer Tool to ensure that primer and probe sets lack hairpins, and homo- and heterodimer interactions. From there, you can link directly to BLAST to further analyze possible sequence interactions. For help with using the BLAST tool, see Tips for using BLAST to locate PCR primers.
2. Use a unique reporter dye to identify each target. Determine the dyes for which your qPCR instrument has been calibrated or is capable of detecting once calibrated. The manufacturer can provide instrument excitation and detectable emission wavelengths. Choose dyes with appropriate excitation wavelengths with little to no overlap in their emission spectra (Table 1, below). Take into account overall fluorescence intensity as well. For example, FAM is a good dye choice for low copy transcripts because it has high fluorescent signal intensity. Fluorophores with lower signal intensities can then be used for more abundant transcripts. Keep in mind that you may need to calibrate your instrument for a particular dye prior to use.
3. Minimize signal cross-talk by using probes that quench well. Highly efficient, dark quenchers—especially those used in combination with a secondary quencher such as the ZEN™ Quencher have an added advantage in multiplex reactions. They considerably reduce background fluorescence leading to increased sensitivity and end-point signal, as well as earlier C_q values.
4. Optimize individual reactions. Ensure that each individual assay reaction is >90% efficient. Test this by running individual assay reactions even if you are using assays that have been published or tested in other laboratories. IDT dsDNA gBlocks™ Gene Fragments and long, ssDNA Ultramer™ DNA Oligonucleotides are ideal as targets for such studies. Validate the multiplex reactions. Run a combined reaction alongside individual reactions to ensure comparable performance. Compare the standard curves and verify that the C_q values are similar throughout the dynamic range to be tested. While the endpoint fluorescent signal will likely be reduced in a multiplex reaction compared to a singleplex, the C_q value should not be affected (Figure 2, above). Optimize the multiplex reactions.

Cy is a registered trademark of Cytiva. ATTO is a trademark of ATTO-TEC GmbH. Texas Red is a registered trademark of Molecular Probes/Life Technologies. JOE is a trademark of Thermo Fisher.

Master mix. The effects of number of targets, target abundance, and amplicon length on the consumption of reaction components are greater for multiplex reactions than singleplex. If any reaction components are limiting, multiplex reactions can show either significant C_q delays in the case of qPCR, or total loss of PCR products, especially for the targets of lowest abundance.

Master mixes specifically formulated for multiplexing are available commercially. When a homemade master mix optimized for singleplex qPCR is being adapted for multiplex reactions, give careful consideration to adjusting the concentration of reaction components. A multiplex reaction will require at least twice as much polymerase as the minimum amount previously titrated for a singleplex qPCR. Additionally, if dNTPs were not in excess for the singleplex reaction, the dNTP concentration will have to be increased. The increased total amount of nucleic acid in the reaction (e.g., from adding additional dNTPs as well as primers/probe, or by generating more template) will decrease the free Mg²⁺ available for the polymerase, requiring adjustment to the Mg²⁺ concentration.

Primer ratio. If the standard 2:1 primer-to-probe ratio for each of the genes analyzed does not provide optimal results, adjust the primer concentrations. For highly expressed targets, use a 1:1 primer-to-probe ratio. Increase the primer-to-probe ratio for targets expressed at lower levels. IDT offers custom primer-to-probe ratio options for PrimeTime qPCR Assays.