The experimental design for multiplex qPCR is more complicated than for single reactions. The probes used to detect individual targets must contain unique reporter dyes with distinct spectra. The settings for excitation and emission filters of real-time detection systems vary from manufacturer to manufacturer; therefore, instruments must be calibrated for each dye as part of the experiment optimization process. Ensuring that instruments are appropriately calibrated will enhance dye specificity, and minimize background and overlap of fluorescent signals.
When selecting dyes:
- Choose dyes that are compatible with your instrument. The instrument must be capable of detecting the emission spectrum for each dye you are using. The manufacturer can provide instrument excitation and detectable emission wavelengths. Get more information on dye spectral emission and excitation values.
- Choose dyes that can be calibrated on your instrument. Your real-time PCR instrument must be calibrated for the set of dyes you choose (Table 1). If it is not, you must calibrate the instrument or select different dyes. IDT provides free, online dye calibration protocols for some common real-time PCR instruments.
- Avoid overlap of emission spectra. Choose dyes with appropriate excitation wavelengths and little to no overlap in their emission spectra (Table 1). Consider total fluorescence intensity as well. For example, FAM is a good dye choice to detect low copy transcripts because it has high fluorescent signal intensity. You can then use fluorophores with lower signal intensities for more abundant transcripts (e.g., housekeeping genes).
Table 1. Dye recommendations for multiplex qPCR. Shown are up to 5 pre-calibrated dye combinations for common real-time PCR instruments. Some machines (most of the Applied Biosystems instruments) will require calibration for HEX and JOE dyes. Also, additional dyes are available from IDT, but may require prior dye calibration. You can access free, online calibration protocols for some of the common real-time PCR instruments.
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 (Table 2), considerably reduce background fluorescence leading to increased sensitivity and end-point signal, as well as earlier Cq values (see the data in the article, Decrease qPCR Background, Improve qPCR Signal). This is particularly useful for multiplex reactions because having several fluorophores in the same tube causes higher background fluorescence. We recommend using the same “quencher type” (all dark quenchers or all fluorescent quenchers) in assays that will be multiplexed.
Table 2. Suggested quenchers for probes used in multiplex qPCR. Note that you must verify that your qPCR instrument is calibrated for the dyes you choose, and if not, you must perform calibration or select different dyes. Read more about dye spectral emission and excitation values.
1 NHS ester
2 The TEX™ 615 spectrum overlaps with that of ROX; therefore, TEX 615 should not be used in a platform that requires a ROX passive reference or with master mixes containing high concentrations of ROX dye.