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A typical real-time PCR assay includes two primers and, when using Applied Biosystems\u2122 TaqMan\u2122 chemistry, a fluorescently labeled probe. In singleplex experiments, one assay is amplified per well. In multiplex qPCR, two or more targets are amplified simultaneously, each identified by a probe labeled with its own dye.
Among available chemistries, Applied Biosystems\u2122 TaqMan\u2122 probe\u2013based assays are among the best suited for multiplexing because each probe provides both specificity and independent fluorescence detection. This dual capability allows researchers to amplify multiple assays in one reaction and detect each target independently.<\/p>\n\n\n\n
Why choose multiplex qPCR?<\/h2>\n\n\n\n
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Multiplex qPCR can offer important advantages over singleplex workflows:
\u2022 Higher throughput \u2013 Detect more targets per well, increasing the number of samples that can be amplified in a plate.
\u2022 Lower reagent and sample use \u2013 Efficiently use reagents and limited biological samples.
\u2022 Improved precision and reliability \u2013 Internal controls within the same well normalize variability.
\u2022 Enhanced data confidence \u2013 Unlike singleplex experiments, all targets and the control are tied to each other in each well.
While duplexing (two targets per well) is often a practical first step, the power of multiplex qPCR can be further increased by combining three or more assays in a single reaction. Thermo Fisher Scientific’s advanced tools, services and products make high-order multiplexing (up to six targets) not only achievable but increasingly accessible.<\/p>\n\n\n\n
When multiplexing makes the most sense<\/h2>\n\n\n\n
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Multiplexing helps deliver the most significant benefit when you are:
\u2022 Amplifying large sample cohorts using a consistent set of assays.
\u2022 Needing more quality assurance in the data.
\u2022 Working with limited or precious sample material.
If your project requires a large number of assays (for example, more than 12), a real-time PCR array may be more efficient.
As the number of targets in the multiplex increases (3\u20136), careful planning becomes even more important.\u00a0 Thermo Fisher Technical Applications Scientists and Field Applications Scientists can provide guidance.<\/p>\n\n\n\n
How to design a successful multiplex qPCR assay<\/h2>\n\n\n\n
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The recipe for developing an effective multiplex qPCR workflow includes assay considerations and testing.<\/p>\n\n\n\n
1. Identify compatible assays<\/strong><\/h3>\n\n\n\nStart by selecting the assays you wish to combine. The number of assays you can multiplex depends on your instrument\u2019s optical filters. For instance, if your real-time PCR instrument has six detection channels, you can theoretically multiplex up to six targets.
Use the Multiplexing Support Request Tool<\/a> on the qPCR Assay Design Hub<\/a> to check assay compatibility. Thermo Fisher\u2019s Multiplexing Support Team offers free in silico<\/em> analysis to identify potential unwanted primer or probe interactions.<\/p>\n\n\n\n2. Choose the right reporter dyes<\/strong><\/h3>\n\n\n\nProper dye selection helps ensure clear spectral discrimination. Recommended dyes include: Applied Biosystems\u2122 FAM\u2122, VIC\u2122, ABY\u2122\/NED\u2122, JUN\u2122, Cy5\u2122, and Cy5.5\u2122. Most Applied Biosystems\u2122 reagents include the passive reference dye ROX\u2122, which provides many benefits \u2014 especially for multiplex reactions. Consider avoiding red reporter dyes, such as JUN, to allow the use of ROX.<\/p>\n\n\n\n
ABY, NED, and TAMRA are all orange-emitting dyes. Avoid having multiple dyes of similar emissions in a multiplex. Using dyes, the instrument is already calibrated for, simplifies multiplexing. When using non-standard dyes, called custom dyes, custom dye calibrations will be needed.<\/p>\n\n\n\n
3. Primer concentrations<\/strong><\/h3>\n\n\n\nFor singleplex TaqMan probe-based assays, universal primer concentration (900 nM) is recommended. However, in a multiplex, universal primer concentration could allow a more abundant target\u2019s amplicon to reach a concentration that saturates Taq<\/em> DNA polymerase, which could suppress amplification of lesser abundant targets. To prevent polymerase saturation in a multiplex, an assay can be formulated with a reduced primer concentration, called primer-limited. The researcher should choose which assays need to be primer-limited based on anticipated target abundances.<\/p>\n\n\n\n![\"Diagram](\"https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2025\/12\/Example-of-polymerase-saturation-1024x777.jpg\")
Example of polymerase saturation<\/strong><\/figcaption><\/figure>\n\n\n\nIf universal singleplex primer concentration (900nM) is used, the higher abundance gene continues to amplify until the polymerase is saturated, causing amplification to transition to linear phase. Amplification of the lower abundance gene must also transition to linear phase when Taq<\/em> DNA polymerase is saturated. In this saturation scenario, the Ct value of the lesser abundant gene is inaccurate or even absent; the multiplex failed.<\/p>\n\n\n\n![\"Graph](\"https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2025\/12\/Higher-abundance-gene-1024x512.jpg\")
<\/figure>\n\n\n\n<\/p>\n\n\n\n
Polymerase saturation is prevented by a reduction in primer concentration, called \u201cprimer limitation.\u201d A primer limited assay transitions from exponential to linear phase due to primer exhaustion rather than saturation of the polymerase, allowing exponential amplification to be detected for the lower abundance gene.<\/p>\n\n\n\n
VIC-labeled, primer-limited (PL)<\/a> formats can be selected online for Applied Biosystems\u2122 TaqMan\u2122 \u00a0Gene Expression Assays<\/a>\u00a0or Applied Biosystems\u2122 Custom TaqMan\u2122 \u00a0Gene Expression Assays.\u00a0 Specialty oligos<\/a> can also synthesize assays with different reporter dyes and primer-limited format.
<\/a><\/p>\n\n\n\n4. Performance testing before scale-up<\/strong><\/h3>\n\n\n\nBefore using a multiplex for its intended application, its performance should be tested. The more rigorous method of performance testing is by mixed standard curves. One target is serially diluted across a range likely to be encountered in samples. To these serial dilutions are added a consistent amount of a second target. The mixed standard curve is amplified in real-time PCR. Ideally, both targets will maintain linearity in the standard curve plot across the entire range tested. If the multiplex will be triplex or higher, multiple pairs of targets will need to be tested in this way. Note that Invitrogen\u2122 GeneArt\u2122, a part of Thermo Fisher, can synthesize DNA controls needed to create mixed standard curves.<\/p>\n\n\n\n
A less rigorous way to test the performance of a multiplex is to amplify a representative collection of samples in singleplex and multiplex, and compare the results.<\/p>\n\n\n\n
5. Synthesize multiplex assay into one tube (optional)<\/strong><\/h3>\n\n\n\nIf the performance of the multiplex has been verified as being acceptable, specialty oligos can help deliver the assays in a single tube format, which simplifies preparing the reactions and helps ensure consistent ratios among all the primers and probes.
<\/p>\n\n\n\n
For Research Use Only. Not for use in diagnostic procedures<\/p>\n\n\n\n
<\/p>\n\n\n\n
Need expert help? Contact us<\/h2>\n\n\n\n
\n\n\n\n
Ready to streamline your workflow with multiplex qPCR?<\/p>\n\n\n\n
\ud83d\udcde Call 800-955-6288, option 2. When prompted, say \u201ctechnical support,\u201d then specify \u201creal-time PCR.\u201d<\/p>\n\n\n\n
<\/p>\n\n\n\n
Thermo Fisher\u2019s Technical Applications Scientists and Field Applications Scientists are ready to help you design, test, and optimize your multiplex qPCR assays. Summary Multiplex qPCR helps you get more results from every reaction by enabling simultaneous detection of multiple targets within a single well. With smart dye and primer concentration selections, you can achieve multiplexing success: efficiently using reagents, time, and sample material. Leverage Thermo Fisher Scientific\u2019s tools, resources, and experienced professional guidance to bring your multiplex qPCR experiments to life.
<\/p>\n\n\n\n
Explore: Multiplexing resources<\/a> | Multiplexing Support Request Tool<\/a> | Predesigned TaqMan\u2122 Assays<\/a><\/p>\n\n\n\n<\/p>\n\n\n\n
\u00a9 2025 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license.<\/p>\n\n\n\n
Use the Multiplexing Support Request Tool<\/a> on the qPCR Assay Design Hub<\/a> to check assay compatibility. Thermo Fisher\u2019s Multiplexing Support Team offers free in silico<\/em> analysis to identify potential unwanted primer or probe interactions.<\/p>\n\n\n\n
2. Choose the right reporter dyes<\/strong><\/h3>\n\n\n\nProper dye selection helps ensure clear spectral discrimination. Recommended dyes include: Applied Biosystems\u2122 FAM\u2122, VIC\u2122, ABY\u2122\/NED\u2122, JUN\u2122, Cy5\u2122, and Cy5.5\u2122. Most Applied Biosystems\u2122 reagents include the passive reference dye ROX\u2122, which provides many benefits \u2014 especially for multiplex reactions. Consider avoiding red reporter dyes, such as JUN, to allow the use of ROX.<\/p>\n\n\n\n
ABY, NED, and TAMRA are all orange-emitting dyes. Avoid having multiple dyes of similar emissions in a multiplex. Using dyes, the instrument is already calibrated for, simplifies multiplexing. When using non-standard dyes, called custom dyes, custom dye calibrations will be needed.<\/p>\n\n\n\n
3. Primer concentrations<\/strong><\/h3>\n\n\n\nFor singleplex TaqMan probe-based assays, universal primer concentration (900 nM) is recommended. However, in a multiplex, universal primer concentration could allow a more abundant target\u2019s amplicon to reach a concentration that saturates Taq<\/em> DNA polymerase, which could suppress amplification of lesser abundant targets. To prevent polymerase saturation in a multiplex, an assay can be formulated with a reduced primer concentration, called primer-limited. The researcher should choose which assays need to be primer-limited based on anticipated target abundances.<\/p>\n\n\n\nExample of polymerase saturation<\/strong><\/figcaption><\/figure>\n\n\n\nIf universal singleplex primer concentration (900nM) is used, the higher abundance gene continues to amplify until the polymerase is saturated, causing amplification to transition to linear phase. Amplification of the lower abundance gene must also transition to linear phase when Taq<\/em> DNA polymerase is saturated. In this saturation scenario, the Ct value of the lesser abundant gene is inaccurate or even absent; the multiplex failed.<\/p>\n\n\n\n<\/figure>\n\n\n\n<\/p>\n\n\n\n
Polymerase saturation is prevented by a reduction in primer concentration, called \u201cprimer limitation.\u201d A primer limited assay transitions from exponential to linear phase due to primer exhaustion rather than saturation of the polymerase, allowing exponential amplification to be detected for the lower abundance gene.<\/p>\n\n\n\n
VIC-labeled, primer-limited (PL)<\/a> formats can be selected online for Applied Biosystems\u2122 TaqMan\u2122 \u00a0Gene Expression Assays<\/a>\u00a0or Applied Biosystems\u2122 Custom TaqMan\u2122 \u00a0Gene Expression Assays.\u00a0 Specialty oligos<\/a> can also synthesize assays with different reporter dyes and primer-limited format.
<\/a><\/p>\n\n\n\n4. Performance testing before scale-up<\/strong><\/h3>\n\n\n\nBefore using a multiplex for its intended application, its performance should be tested. The more rigorous method of performance testing is by mixed standard curves. One target is serially diluted across a range likely to be encountered in samples. To these serial dilutions are added a consistent amount of a second target. The mixed standard curve is amplified in real-time PCR. Ideally, both targets will maintain linearity in the standard curve plot across the entire range tested. If the multiplex will be triplex or higher, multiple pairs of targets will need to be tested in this way. Note that Invitrogen\u2122 GeneArt\u2122, a part of Thermo Fisher, can synthesize DNA controls needed to create mixed standard curves.<\/p>\n\n\n\n
A less rigorous way to test the performance of a multiplex is to amplify a representative collection of samples in singleplex and multiplex, and compare the results.<\/p>\n\n\n\n
5. Synthesize multiplex assay into one tube (optional)<\/strong><\/h3>\n\n\n\nIf the performance of the multiplex has been verified as being acceptable, specialty oligos can help deliver the assays in a single tube format, which simplifies preparing the reactions and helps ensure consistent ratios among all the primers and probes.
<\/p>\n\n\n\n
For Research Use Only. Not for use in diagnostic procedures<\/p>\n\n\n\n
<\/p>\n\n\n\n
Need expert help? Contact us<\/h2>\n\n\n\n
\n\n\n\n
Ready to streamline your workflow with multiplex qPCR?<\/p>\n\n\n\n
\ud83d\udcde Call 800-955-6288, option 2. When prompted, say \u201ctechnical support,\u201d then specify \u201creal-time PCR.\u201d<\/p>\n\n\n\n
<\/p>\n\n\n\n
Thermo Fisher\u2019s Technical Applications Scientists and Field Applications Scientists are ready to help you design, test, and optimize your multiplex qPCR assays. Summary Multiplex qPCR helps you get more results from every reaction by enabling simultaneous detection of multiple targets within a single well. With smart dye and primer concentration selections, you can achieve multiplexing success: efficiently using reagents, time, and sample material. Leverage Thermo Fisher Scientific\u2019s tools, resources, and experienced professional guidance to bring your multiplex qPCR experiments to life.
<\/p>\n\n\n\n
Explore: Multiplexing resources<\/a> | Multiplexing Support Request Tool<\/a> | Predesigned TaqMan\u2122 Assays<\/a><\/p>\n\n\n\n<\/p>\n\n\n\n
\u00a9 2025 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license.<\/p>\n\n\n\n
For singleplex TaqMan probe-based assays, universal primer concentration (900 nM) is recommended. However, in a multiplex, universal primer concentration could allow a more abundant target\u2019s amplicon to reach a concentration that saturates Taq<\/em> DNA polymerase, which could suppress amplification of lesser abundant targets. To prevent polymerase saturation in a multiplex, an assay can be formulated with a reduced primer concentration, called primer-limited. The researcher should choose which assays need to be primer-limited based on anticipated target abundances.<\/p>\n\n\n\n If universal singleplex primer concentration (900nM) is used, the higher abundance gene continues to amplify until the polymerase is saturated, causing amplification to transition to linear phase. Amplification of the lower abundance gene must also transition to linear phase when Taq<\/em> DNA polymerase is saturated. In this saturation scenario, the Ct value of the lesser abundant gene is inaccurate or even absent; the multiplex failed.<\/p>\n\n\n\n <\/p>\n\n\n\n Polymerase saturation is prevented by a reduction in primer concentration, called \u201cprimer limitation.\u201d A primer limited assay transitions from exponential to linear phase due to primer exhaustion rather than saturation of the polymerase, allowing exponential amplification to be detected for the lower abundance gene.<\/p>\n\n\n\n VIC-labeled, primer-limited (PL)<\/a> formats can be selected online for Applied Biosystems\u2122 TaqMan\u2122 \u00a0Gene Expression Assays<\/a>\u00a0or Applied Biosystems\u2122 Custom TaqMan\u2122 \u00a0Gene Expression Assays.\u00a0 Specialty oligos<\/a> can also synthesize assays with different reporter dyes and primer-limited format. Before using a multiplex for its intended application, its performance should be tested. The more rigorous method of performance testing is by mixed standard curves. One target is serially diluted across a range likely to be encountered in samples. To these serial dilutions are added a consistent amount of a second target. The mixed standard curve is amplified in real-time PCR. Ideally, both targets will maintain linearity in the standard curve plot across the entire range tested. If the multiplex will be triplex or higher, multiple pairs of targets will need to be tested in this way. Note that Invitrogen\u2122 GeneArt\u2122, a part of Thermo Fisher, can synthesize DNA controls needed to create mixed standard curves.<\/p>\n\n\n\n A less rigorous way to test the performance of a multiplex is to amplify a representative collection of samples in singleplex and multiplex, and compare the results.<\/p>\n\n\n\n If the performance of the multiplex has been verified as being acceptable, specialty oligos can help deliver the assays in a single tube format, which simplifies preparing the reactions and helps ensure consistent ratios among all the primers and probes. For Research Use Only. Not for use in diagnostic procedures<\/p>\n\n\n\n <\/p>\n\n\n\n Ready to streamline your workflow with multiplex qPCR?<\/p>\n\n\n\n \ud83d\udcde Call 800-955-6288, option 2. When prompted, say \u201ctechnical support,\u201d then specify \u201creal-time PCR.\u201d<\/p>\n\n\n\n <\/p>\n\n\n\n Thermo Fisher\u2019s Technical Applications Scientists and Field Applications Scientists are ready to help you design, test, and optimize your multiplex qPCR assays. Summary Multiplex qPCR helps you get more results from every reaction by enabling simultaneous detection of multiple targets within a single well. With smart dye and primer concentration selections, you can achieve multiplexing success: efficiently using reagents, time, and sample material. Leverage Thermo Fisher Scientific\u2019s tools, resources, and experienced professional guidance to bring your multiplex qPCR experiments to life. Explore: Multiplexing resources<\/a> | Multiplexing Support Request Tool<\/a> | Predesigned TaqMan\u2122 Assays<\/a><\/p>\n\n\n\n <\/p>\n\n\n\n \u00a9 2025 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license.<\/p>\n\n\n\n<\/figure>\n\n\n\n
<\/a><\/p>\n\n\n\n4. Performance testing before scale-up<\/strong><\/h3>\n\n\n\n
5. Synthesize multiplex assay into one tube (optional)<\/strong><\/h3>\n\n\n\n
<\/p>\n\n\n\nNeed expert help? Contact us<\/h2>\n\n\n\n
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<\/p>\n\n\n\n