ROX (5-Carboxy-Rhodamin-X) is a fluorescence dye which some qPCR cyclers use as passive reference dye. This allows to mathematically eliminate non-specific fluorescence signals, resulting from well-to-well variances due to material inconsistencies or unprecise pipetting. So please use this normalisation option if your qPCR machine works with ROX and check which ROX level it requires. However, it is not a problem, if the ROX option is not available. If your qPCR assay is thoroughly validated, you will also get good results without ROX.

Please look here for a list of qPCR cyclers that require ROX.