AAT Bioquest

ROX Reference Dye for Real-Time PCR

qPCR plot
qPCR Plot [Delta Rn = (reporter signal/reference dye signal) - baseline]
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.

Improve the Accuracy and Reproducibility of Your qPCR Results

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:
  • Normalize for non-PCR related fluorescence signal variations due to pipetting inaccuracies, sample effects, bubbling in the wells or well position
  • Normalize for fluorescence fluctuations (e.g. machine noise)
  • Create a stable baseline in multiplex qPCR assays

Table 1. Properties of ROX Reference Dye for qPCR

DyeROX Reference Dye
Concentration50X fluorescence reference solution
ApplicationsReal-time quantitative PCR (qPCR)
Ex/Em (nm)578/604
ε82,000 cm-1M-1
InstrumentsABI PRISM® 7000, 7700 and 7900
Applied Biosystems® 7300 and 7500

Table 2. Ordering ROX Reference Dyes

Ex (nm)
Em (nm)
Unit Size
Cat No.
ROX Reference Dye *50X fluorescence reference solution for PCR reactions*qPCR5786045 mL400
6-ROXtra™ fluorescence reference solution *25 uM for PCR reactions*qPCR5785955 mL398


Additional Resources


Table 3. Possible ROX Reference and reporter dye combinations for multiplex qPCR assays.

Reference Dye
Reporter Dye 1
Reporter Dye 2
Reporter Dye 3
Reporter Dye 4
ABI PRISM® 7000 and 7900
Applied Biosystems® 7300
Applied Biosystems® 7500ROX6-FAM6-TET6-HEXTide Fluor™ 3
iFluor® 647
Alexa Fluor 647