Perfect amplification curves follow a predictable pattern, but anomalies can appear in all sorts of strange shapes. One common issue, especially with ABI instruments, is curves with sharp upward spikes. The reason? Incorrect ROX selection. So, what is ROX, what does it do, why does it cause these curve irregularities, and how can we fix them? Let’s break it down!
What Is ROX?
ROX™ dye, introduced by Applied Biosystems in the 1990s, is an independent, inert dye that doesn’t interfere with the fluorescence of reporter dyes used in real-time PCR (qPCR). During qPCR experiments, factors like pipetting errors, evaporation, condensation, or bubbles can distort fluorescence signals and affect results. ROX™ helps by diagnosing issues such as evaporation, pipetting inaccuracies, or the presence of droplets. It also normalizes abnormal signals caused by bubbles, though it can’t fix poor data quality or correct pipetting errors.
Figure 1. Dynamic Range Comparison of qPCR Amplification on Bio‑Rad CFX384™ vs. Life Technologies ViiA™7 Systems Without ROX™ Dye Normalization
Figure 2. Dynamic range on Life TechnelogiesViiA™ 7system,with Rox™ dye normalization
- Before ROX correction：STD=0.028
- After ROX correction：STD=0.01
Why Do Some Instruments Need ROX Correction While Others Don’t?
It all depends on the instrument’s optical system. For example, Bio-Rad instruments use a uniform light source that evenly illuminates all wells, eliminating optical path differences between them. In this case, the SYBR Green signal directly reflects the fluorescence of the PCR product (see Figure A). On the other hand, ABI instruments typically require ROX correction because their light source varies across wells, creating optical path differences. ROX normalization adjusts for these variations to provide accurate Rn values (see Figure B).
High ROX vs. Low ROX
Instruments requiring High ROX use a blue light source, which strongly excites SYBR Green but weakly excites ROX. This means a higher ROX concentration is needed for proper correction. Low ROX instruments, however, use a mixed blue and white light source—blue light excites SYBR Green, while white light excites ROX—allowing a lower ROX concentration to work effectively.
What If You Choose the Wrong ROX?
If you’ve already run your experiment and notice abnormal data, don’t panic! In the plate setup, simply switch ROX to “none,” reanalyze the data, and you’ll likely get normal amplification curves. If you catch the mismatch before running—say, the qPCR mix doesn’t match your instrument—reach out to your reagent supplier’s technical support to ask about replacements. Even better, modern qPCR reagents now include “universal ROX,” designed to adapt to most instrument models, making life easier for labs with multiple machines.