For certain real-time cyclers, the presence of ROX reference dye in real-time PCR compensates for non-PCR-related variations in fluorescence detection. Fluorescence from ROX reference dye does not change during the course of real-time PCR, but provides a stable baseline to which PCR-related fluorescent signals are normalized. Thus, ROX dye compensates for differences in fluorescence detection between wells due to slight variations in reaction volume or to differences in well position. The use of ROX dye is necessary for all instruments from Applied Biosystems and is optional for the Mx3000P, Mx3005P, and Mx4000®. Instruments from Bio-Rad/MJ Research, Cepheid, Corbett Research, Eppendorf, and Roche do not require ROX dye.