ROX Reference Dye
Some real-time instruments recommend the use of a passive reference dye (typically ROX) to overcome well-to-well variations that could be caused by machine limitations such as “edge effect”, bubbles, small differences in volume, and autofluorescence from dust or particulates in the reaction. However, ROX normalization does little to the variations caused by pipetting errors of templates/primers, heterogeneous mixing, and evaporation/condensation issues.
A universal passive reference dye is included in the following Luna® qPCR products: Luna Universal qPCR Master Mix (NEB #M3003), Luna Universal Probe qPCR Master Mix (NEB #M3004), Luna Universal One-Step RT-qPCR Kit (NEB #E3005), and Luna Universal Probe One-Step RT-qPCR Kit (NEB #E3006). These products support broad instrument compatibility (High-ROX, Low-ROX, ROX-independent) so no additional ROX is required for normalization.
The Luna Probe One-Step RT-qPCR Kit (No ROX) (E3007) contains no reference dye and is compatible with any instrument that does not require ROX. If ROX normalization is needed, ROX can be added. Please refer to instrument manufacturer’s instructions for greater details.