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.
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 free online software 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.
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. 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, have an added advantage in multiplex reactions. They considerably reduce background fluorescence leading to increased sensitivity and end-point signal, as well as earlier Cq 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.
5. Validate the multiplex reactions. Run a combined reaction alongside individual reactions to ensure comparable performance. Compare the standard curves and verify that the Cq 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 Cq value should not be affected.
6. Optimize the multiplex reactions:
- 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 Cq 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.