TaqMan™ Genexpressionsassay, VIC

Yes, it is possible. The amplicons do not have to be the same length in order to duplex the assays. You need to use different reporter dyes on the probes in multiplex - they should have a substantial difference in maximum emission wavelength to be effective. A classic example is to use FAM and VIC for the 2 probes. Depending on the expression of the targets, you may also need to limit the primer concentration for one of the assays. This is common when duplexing a target and endogenous control (the control probe is VIC labeled and the primer concentration is decreased so the target assay can properly compete for use of enzyme and dNTPs).

You can utilize the Applied Biosystems TaqMan Multiplex Interaction Check (MIC) tool. The MIC tool uses bioinformatics to identify potential interactions between primers, probes, and amplicons that could negatively impact multiplex qPCR performance. The tool was developed and validated using data from approximately 200 multiplex panels, combining computational analysis with wet-lab testing. By flagging assay combinations that may be prone to false positives or reduced sensitivity, MIC tool helps researchers design multiplex panels with greater confidence and reduce time spent on trial-and-error optimization.risk and observed multiplex performance.

Primer-Limited Applied Biosystems TaqMan Gene Expression Assays with FAM, ABY, JUN, or Cyanine 5 dyes have a primer concentration of 350 nM. Assays with VIC dyes have a primer concentration of 150 nM.

In a multiplex qPCR reaction, like in Applied Biosystems TaqMan Gene Expression Assays, targets often differ in abundance and amplification efficiency. Highly expressed or highly efficient targets can consume a disproportionate share of reaction components, generate very strong fluorescent signals, and outcompete weaker targets for polymerase and other reagents. Primer-limited assays provide balanced amplification of targets in multiplex workflows by reducing primer competition between assays and supporting more consistent amplification across multiple targets.

You can multiplex up to five Applied Biosystems TaqMan Gene Expression Assays utilizing FAM, VIC, ABY, JUN, and Cyanine 5 dyes.