6-carboxy-X-rhodamine. Most commonly used passive reference dye for normalization of reporter signals in ABI instruments. The emission recorded from ROX during the baseline cycles (usually 3 to 15) is used to normalize the emission recorded from the reporter due to amplification in later cycles. The use of ROX improves the results by compensating for small fluorescent fluctuations such as bubbles and well-to-well variations that may occur in the plate. Not using ROX or not designating it as the passive reference dye in the analysis may cause trailing of the clusters in the allelic discrimination plot if the instrument (like the ABI) requires a passive reference. ROX or any other internal reference dye is not required by all machines (see the list in Table 1 in Qiagen Publication: Checklist for Multiplex Real-time PCR). If in a ROX requiring instrument, a master mix with lower than required ROX concentration is used, the SD will be large and may be reduced by using an appropriate master mix.