ROX is a fluorescent molecule that the real-time PCR system can detect when its present in the reaction. It’s used as a Passive reference dye for normalization of fluorescence signal across all of the PCR samples of the PCR thermo cycler including a baseline.
Rox is required when there is uneven illumination, sample variation and difference in quantity or condensation.
Its shadowing the reporter as a constant fluorescent and results in a higher precision of well data.
Adding ROX depends on the Real-time PCR instrument you’re using. Companies such as for example Bioline offer an easy selection tool on their website, informing you about the exact requirements for your instrument.