ROX dye is applied as a passive reference dye when using a real-time PCR systems to reduce the fluorescence fluctuations from well to well. Ref. dye provides a constant level of fluorescence in each well during the reaction. This dye does not affect the amount of DNA in the subsequent calculation. However, you can not use the optical channel occupied by ROX in multiplex PCR.
Add necessary volume of ROX Reference Dye to master mix (depends on PCR machine type).
Different types of real-time PCR systems that require a passive reference standard have different optimal concentrations of ROX reference, mainly due to the different optical configurations of each system, particularly the type of excitation source and optics used.