SuperRox is a specially formulated version of the ROX (carboxy-X-rhodamine) fluorophore. This dye is commonly used as passive reference to normalize signal variations not related to the PCR amplification. Irregularities controlled with a ROX passive reference include inconsistency in pipetting, evaporation of solution, instability of baseline fluorescence, and laser or light source anomalies which all produce well-to-well variation.
SuperROX is formulated for maximum water solubility so as to guard against “ROX drop”, a linear decrease in ROX signal occurring throughout the PCR reaction. Presumably, this drop is due to aggregation of hydrophobic ROX molecules into intermolecular homodimers, accompanied by “static quenching” of the fluorescent signal. SuperROX is an ancillary reagent that does not participate in the PCR reaction. The SuperROX signal remains consistent throughout the experiment thus providing a stable reference to correct the baseline fluorescence and normalize the amplified signal.
SuperROX is for use only in machines requiring a passive reference dye and with optics capable of resolving the ROX fluorophore (Abs Max 585 nm / Em Max 606 nm). SuperROX is typically utilized at concentrations between 100 and 300 nanomolar in the final reaction mix. Further concentration optimization may be necessary on certain model instrumentation.