AriaMx HEX-JOE and CY3 Cross-Talk

Answer

This Information Applies To: Agilent AriaMx real-time PCR system, AriaMx real-time PCR optical modules

Issue

Users may observe an unexpected fluorescence signal in the CY3 channel when using the fluorophore HEX or JOE. This issue can lead to misinterpretation of data if not properly managed.

Background

The root cause of this phenomenon is "cross-talk" between the HEX-JOE and CY3 optical modules. Cross-talk occurs when two different optical modules (the target optical module and the spillover optical module) detect the emission from one dye. A dye is at risk for cross-talk when its emission spectra overlaps that of another dye that is assigned to a different optical module. Figure 1 shows the overlapping excitation and emission spectra of HEX, JOE, and Cy3.

Figure 1. Excitation and emission spectra for the fluorescent dyes JOE, HEX, and Cy3

Caution: The spectral proximity of JOE, HEX, and Cy3 is such that their signals are indistinguishable when using standard detection methods. Agilent does not recommend using HEX, JOE, and Cy3 together in a multiplex reaction.

Tip: The factory settings for cross-talk correction have been optimized to eliminate potential cross-talk for dyes that are part of the default optical configuration. Agilent does not recommend changing the cross-talk correction settings unless you are using a new custom dye and more than one optical module could detect the emission wavelength. How to change the default cross-talk settings is explained in chapter 2 of the Aria Real-Time PCR Software User Manual.

Resolution

Cross-talk can be avoided by choosing dyes for multiplexing experiments that have clearly separate excitation and emission spectra, e.g. HEX and FAM, or HEX and ATTO 425

The optical module excitation and emission wavelengths are detailed in the table in the photo roll on the AriaMx product page, as well as in Table 1. To determine whether the fluorescence dye of interest might work, you can use a spectral overlay tool. 

Table 1. AriaMx optical module excitation and emission wavelengths

Tip: To determine which fluorescent dyes are compatible with your optical modules, see the article Which Fluorescent Dyes Can Be Detected with the AriaMx?

Learn how to effectively operate your Agilent AriaMx Real-Time PCR System: AriaMx Real-Time PCR System Setup and User Guide on Agilent.com.  Aria Real-Time PCR Software User Manual on Agilent.com.

For Research Use Only. Not for use in diagnostic procedures.

PR7001-2451