What is multiplex qPCR?

Let’s suppose you use qPCR to determine the expression of oncogenes or tumor suppressor genes in a tumor biopsy sample. These clinical samples are difficult to collect and generally only contain a small amount of usable mRNA.

In a singleplex relative quantification experiment, only one gene—either the gene of interest or the control—is amplified in each well. Assuming that each assay is performed in triplicate, you will need to divide your sample into six wells (three for the test gene and three for the endogenous control) to measure the expression of a single gene. Thus, the number of genes that can be tested using the limited amount of biopsy sample will be restricted by running singleplex reactions for each gene.  However, these limitations can be overcome by amplifying the 2 genes (or multiple genes) in the same reaction.

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The simplest, and most commonly used, type of multiplexing is duplexing, in which two genes are amplified in a single reaction. Typically, in a relative qPCR experiment designed to determine the fold change differences in gene expression, these will be a single gene of interest (target gene) and an endogenous control. TaqMan assays to detect the target and control genes, containing probes that have been labeled with 2 distinct fluorescent dyes, are added to the same reaction, and use the same pool of Taq polymerase enzymes, nucleotides, and other reagents during amplification.

The real time PCR instrument used for performing amplification must be capable of distinguishing precisely between these fluorescent labels and measuring the signals produced by the amplification of each gene. Typically, the probe for your gene of interest is labeled with FAM dye and the probe for the control with VIC dye. The emission spectra for FAM and VIC peak at 517nm (in the blue region of the visible spectrum) and 551nm (in the green region) respectively (Figure 1), making these wavelengths easily distinguishable by any Applied Biosystems real time PCR instrument.

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You can, under carefully optimized conditions, perform multiplex qPCR to measure the expression of three or four genes simultaneously in a reaction. This can provide huge savings in cost, reagents and time, but the resulting experiments are more complex, and validation becomes more time-consuming.  When interrogating 3 or 4 targets in the same well, there is even more competition for shared reagents than in a duplex reaction, and the scope for unwanted interactions between primers and probes increases. Thus it is necessary to validate the multiplex reaction thoroughly following the same guidelines outlined above for performing a duplex reaction.

In a multiplex reaction with 2 or more targets, you can utilize TaqMan assays for the genes of interest, with each assay probe labeled with a different dye.  Applied Biosystems range of dyes now also includes ABY and JUN, with fluorescence spectra that peak at 580 nm (yellow) and 617 nm (orange-red) respectively and can be used in conjunction with FAM and VIC dyes.

The TaqMan quencher, QSY, is available for optimal high-level multiplexing with best performance in 3 and 4-plex reactions. Similar to the MGB-NFQ quenchers, QSY is a non-fluorescent quencher, however the QSY probes don’t have a MGB moiety. If for example, you are running a 4-plex reaction, two TaqMan assays can be FAM and VIC probes labeled with an MGB-NFQ quencher while the other two assays should have ABY and JUN labeled probes with a QSY quencher.

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• Competition or inhibition between assays through interactions among the various primer pairs, probes, targets, amplicons, or any combination.
• The relative expression levels of targets (including endogenous controls), and the dynamic range of their expression.

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Important considerations when optimizing multiplex assays include:

• Primers should be specific and should not be able to bind elsewhere in the template DNA, to the probe, or to each other.
  
  
• The Tm of TaqMan probes should be ~10°C higher than the Tm of the primers (approximately 68–70°C). If you are currently using TaqMan predesigned assays containing MGB-NFQ probes (also referred to as MGB probes), you can continue to use these assays in a multiplex reaction. However, the multiplex reaction should not contain more than two MGB probes to ensure successful amplification. Please note, that MGB probe sequences are not interchangeable with QSY probe sequences because the QSY probe length will be too short. If you are using a TaqMan predesigned assay click here to learn more about our offerings and to have assays redesigned.
  
  
• A multiplex reaction can contain up to eight primers and four probes (to produce four amplicons), so it is good practice to minimize conditions that result in primer dimer formation or other unfavorable interactions. Make sure that amplicons do not overlap. If the amplicon coordinates are not known, map genomic assays to the genome, or gene expression assays to the transcriptome. A web-based tool that can be used in verification of coordinates is the UCSC Genome Browser In Silico PCR utility at: genome.ucsc.edu/cgi-bin/hgPcr. It is also important to make sure that amplicons are all approximately the same size, and that primer and probe dimers do not form (across all primer pairs).  Use our Multiple Primer Analyzer tool to check for primer dimer formation.
  
  
• The ABY and JUN probes must be made with QSY, but FAM and VIC probes can be made with either QSY or MGB. For example, multiplex gene expression analysis can use FAM and VIC TaqMan Gene Expression Assays combined with one assay containing a custom ABY-QSY probe and one assay containing a custom JUN-QSY probe. Also, for multiplex SNP genotyping analysis, an existing FAM/VIC TaqMan SNP Genotyping assay can be combined with a custom ABY-QSY/JUN-QSY assay.
  
  
• Choose dyes with little to no overlap in their emission spectra. Also, match dye intensity with target abundance by pairing the brightest dye with low abundance targets, and the dimmest dye with high abundance targets (e.g., an internal positive control).
  
  
• Because all of the assays are amplified in the same tube, they compete for the same reagents (dNTPs, Mg2+, and polymerase). The more targets that are assayed in a multiplex reaction, the more likely it is that there will be competition for reagents and inhibition between assays. Master mixes specifically designed for performing multiplex PCR should be used to offset the effect of competition for reagents.  .Applied Biosystems TaqMan Multiplex Master Mix, TaqPath 1-Step Multiplex Master Mix*, and TaqPath ProAmp Master Mixes* are all optimized for use with multiplexing reactions and are formulated with Mustang Purple dye as the passive reference dye instead of ROX to accommodate the use of the JUN dye in high target multiplexing.

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The many advantages of multiplex qPCR experiments arise from the fact that the assays for the test and the control gene use the same reagents in the same reaction. These reactions are, however, in competition for the same limited pool of reagents. This could cause problems when one gene (most often the control) is much more abundant than the other gene(s) in the sample. In this case, the highly expressed gene will start amplification earlier in the run than the less abundant gene(s), and may even reach its linear and plateau phases before the less abundant gene(s) has started amplification. The amplification of the first gene may thus use up most of the nucleotides and other reagents in the pool. This leaves the remaining genes without enough reagents to amplify properly, and the Ct value recorded will not reflect actual abundance.

Fortunately, this problem has a simple solution: primer limitation, which involves running the assay for the more abundant gene with strictly limited primer amounts. Under these circumstances, that gene will reach its plateau more quickly due to running out of primers, and not due to the lack of reagents, which are in excess.  There should be enough nucleotides, polymerase and other reagents left for the amplification of the less abundant genes. In a primer limited multiplex reaction, the Ct values for both genes will still be measured accurately.

In a typical singleplex TaqMan reaction the primer concentrations are 900nM each and the probe has a concentration of 250nM.  In a primer limited assay, the primers are typically reduced to 150nM each with the probe concentrations remaining unchanged.

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Multiplexing will not always be appropriate for your qPCR experiments. Before you embark on a multiplex experiment, optimize the assay conditions and validate them carefully.

Confirm that results obtained from multiplexing are the same as results obtained from singleplex reactions. The general procedure is as follows:

1. Run your singleplex reactions and confirm amplification.
2. After establishing conditions for performing singleplex reactions, set up conditions for multiplex reactions. Ensure amplification occurs and analyze data.
3. Determine whether the singleplex and multiplex reactions give the same Ct values.
4. If the singleplex and multiplex reactions do not give the same Ct values, optimize primer/probe concentrations if necessary to obtain the desired ∆Ct.

Each reaction should be carried out in triplicate

As multiplexing minimizes pipetting errors, you might assume that by using this technique, you guarantee low variation in Ct values between replicates. This is not always the case, as variation can arise from interactions between the complex mix of reagents in the well. If the variation between replicates is high, you can try to increase precision by increasing the number of replicates, though this will reduce your reagent and cost savings. Widely varying results suggest you should return to singleplexing.

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1. Henegariu O, Heerema NA, Dlouhy SR et al. (1997) Multiplex PCR: Critical parameters and step-by-step protocol. Biotechniques 23(3):504–511.