Does Open qPCR require a ROX reference dye?

Open qPCR does not require a ROX or any other reference dye.

Some instruments use a reference dye to normalize well-to-well differences. Because of Open qPCR's unique, solid-state optical architecture & calibration algorithms, no reference dye is required.

If you will be using a master mix containing a ROX reference dye, when calibrating your dual channel instrument, it is advisable to reconstitute your FAM + HEX calibrator set with your master mix, rather than Chai's reconstitution buffer solution.