For certain real-time cyclers, ROX passive reference dye is used to compensate for non-PCR-related variations in fluorescence detection. Fluorescence from the ROX reference dye does not change during the course of qPCR, but provides a stable baseline against which PCR-related fluorescent signals are normalized. Thus, ROX compensates for differences in fluorescence detection between wells due to slight variations in reaction volume or differences in well position. The use of ROX (either ROX High or ROX Low) is necessary for all instruments from Applied Biosystems and is optional for the Stratagene Mx series instruments. Instruments from Bio-Rad/MJ Research, Cepheid, Corbett Research, Qiagen, Eppendorf and Roche do not require ROX (although it is important to note that the Bio-Rad iCyclers require fluorescein as a passive reference dye). The presence of ROX in the Master Mix does not interfere with qPCR on any instrument, since the dye is not involved in the reaction and has an emission spectrum different from that of SYBR Green I.