Passive reference dyes, such as the ROX Reference Dye, are essential to the accuracy and reproducibility of real-time PCR (qPCR) reactions performed on ROX-dependent PCR cyclers (e.g. the Applied Biosystems^® instruments). When added to the qPCR master mix, the ROX Reference Dye is designed to normalize the fluorescent signal of qPCR reporter dyes, such as Cyber Green™, Tide Fluor™-Tide Quencher™ modified probes or TaqMan™ probes, and to account for non-PCR related fluorescence signal variations (e.g. pipetting inaccuracies). Since the ROX Reference Dye does not interfere with the qPCR reaction and has an easily discernable red emission spectrum, it provides an excellent baseline in multiplex qPCR assays. Although ROX has been widely used in qPCR, its low stability severely limited its convenience. ROX reference dyes have to be cold-stored to preserve its fluorescence. AAT Bioquest introduced ROXtra™ that has vast improved stability and convenience while preserving the spectral properties of ROX. ROXtra™ reference dye has been steadily used for replacing ROX reference dyes for the qPCR applications that demand speed and convenience, such as the rapid Covid-19 qPCR tests.

The ROX reference dye offers are convenient method for normalizing fluorescent reporter signals in qPCR without having to modify the instrument's default analysis parameters. In addition, the ROX reference dye can act as an internal control to: