FAQ: Do I need to add ROX?

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.

The following Luna® qPCR products contain a universal passive reference dye that enables compatibility with a variety of real-time instruments, including those that use no passive reference normalization and those that use a low or high amount of passive reference dye (ROX).

Luna Universal qPCR Master Mix (NEB #M3003)
Luna Universal Probe qPCR Master Mix (NEB #M3004)
Luna Universal One-Step RT-qPCR Kit (NEB #E3005)
Luna Universal Probe One-Step RT-qPCR Kit (NEB #E3006)
Luna Probe One-Step RT-qPCR 4X Mix with UDG (NEB #M3019)

LyoPrime Luna Probe One-Step RT-qPCR Mix with UDG (NEB #L4001)

The Luna Probe One-Step RT-qPCR Kit (No ROX) (NEB #E3007) ) and Luna Probe One-Step RT-qPCR 4X Mix with UDG (No ROX) (NEB #M3029) lack a reference dye and can be used with any instrument that does not require ROX normalization. Please refer to instrument manufacturer’s instructions for greater details.

qPCR Instrument ROX

Bio-Rad® iQ™5, CFX96, CFX384, Opticon
Roche Lightcycler®
Qiagen Rotor-Gene™
Eppendorf Mastercycler®
Cepheid® SmartCycler®

Not recommended/required

Applied Biosystems® 7500, 7500 Fast, QuantStudio™, ViiA7™ Agilent Mx™

(~50 nM final)

Applied Biosystems® 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne™, StepOnePlus™

High ROX
(~500 nM final)