Between ROX and a Hard Place

Updated : Mon, September 19, 2022 @ 2:10 PM

A "Referral from the Doctor" Blog Article-


Quantitative real-time RT-PCR was developed as a means of quantifying gene expression either relatively or absolutely, with accurate and reproducible results. Despite advances in dye chemistries, variability continues to haunt research scientists.  One effort to reduce the observed variability between technical replicates was to include the ROX dye (5- or 6-carboxy-X-rhodamine) as a passive reference in the PCR master mix.

The role of ROX dye in qPCR is to provide fluorescence normalization and to help overcome equipment limitations. Early versions of real-time PCR machines use a CCD camera image to photograph the fluorescence intensity in all wells. Unfortunately, the recorded intensity can be dependent upon the position of the well as seen in “edge effects” where the outer most row of a 96 well plate provides lower signal than the technical replicates located more centrally. In effect, these outer wells are compromised and the reproducibility of data cast into doubt. The addition of ROX dye provides a baseline level of fluorescence with which all wells can ‘normalized’ for signal intensity. More recent equipment designs may use mirrors to redirect the fluorescent signal to the detector eliminating well position effects. In this regard, it is essential that researchers know which machines require the inclusion of ROX in their study. ROX is required when using the ABI Prism® 7000, 7500 and 7900. ROX is optional for Bio-Rad’s iCycler IQ®, Opticon®, Chromo™ 4, and Strategene’s MX3000P™ or MX4000®. ROX is not required for use of Qiagen's Rotor-Gene™ 6000, and Roche's LightCycler® series. Master mixes from various vendors and equipment manufacturers may be purchased with or without ROX dye added when supplied as a separate component.

Fluorescence Normalization

Optimization of the ROX passive reference dye concentration is an essential component of qPCR data reproducibility and should be determined for each real-time machine model and manufacturer. In ‘normalization’ calculations the fluorescence emission intensity of the reporter dye (FIR) is divided by the fluorescence emission intensity of the passive reference dye (ROX) to obtain a ratio for the normalized reporter or Rn. The difference between the Rn values of the baseline and the template-containing sample equals the delta (Δ)Rn, indicating the magnitude of signal generated by the sample under those specific PCR conditions. Absolute or relative expression is determined on the basis of an assigned threshold and the number of cycles needed to cross that threshold (Ct, Cp or Cq).

ROX drop

When added to the PCR reaction the ROX dye-conjugate does not participate in the 5’-exonuclease-induced signal generation, does not affect reaction efficiency and is thus used as a passive reference. Unfortunately, one characteristic of the commonly used ROX dye conjugates is an observed “ROX drop” in signal intensity throughout thermal cycling. Based on the chemical properties of a traditional ROX passive reference dye it is assumed that the ROX-conjugates are aggregating and the emission signal quenched through static or contact quenching mechanisms. This aggregation may be due to insolubility in the master mix resulting in a progressive decrease in the effective concentration of ROX.

The SuperRox® solution

SuperRox DyeSuperRox is a custom version of the ROX passive reference dye manufactured by Biosearch Technologies, Inc. SuperRox has all the favorable characteristics of other ROX reference dyes with the added feature of water solubility. Without dye aggregation and dye conjugate precipitation the ROX drop incidence is negligible. SuperRox has been shown to maintain a constant signal throughout PCR thermocycling providing an unwavering source for fluorescence baseline normalization. Substituting SuperRox for other ROX reference dyes will increase sample reproducibility, decrease the variability between technical replicates and between repeated experiments.

Written by: Christina Ferrell, Ph.D., Technical Applications Specialist

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