ROX Passive Reference Dye | Thermo Fisher Scientific

Normalize and troubleshoot your qPCR reactions

ROX is a fluorescent passive reference dye with a level of fluorescence that does not change during the PCR process. Normalization of qPCR data using ROX reduces the influence of well-to-well variability such as uneven illumination, slight variation in optics, and differences in the amount of condensation.

Most real-time PCR instruments use ROX to normalize for these and other sources of variability. ROX is the passive reference dye of choice because it does not influence the PCR process and its emission spectrum does not influence those of many other TaqMan reporter and quencher dyes.

Features

Features of ROX Passive Reference Dye include:

Normalizes non-PCR-related fluctuations in fluorescence

Using a passive reference like ROX dye to normalize data helps achieve a higher level of precision among technical replicates. Without normalization, more replicates may be required to achieve comparable precision levels, increasing the time and resources required for each run. Data normalized with ROX dye is simply more precise than data without it, enabling researchers to run fewer replicates while maintaining statistical power.

Provides a stable baseline for multiplex quantitative PCR and RT-PCR

In a multicomponent plot, the ROX signal should remain constant through the qPCR process. Any non-PCR related events will lead to spikes, dips, or other abnormal behavior in the reporter and ROX signals.

Using ROX dye to troubleshoot common qPCR issues

Unlike probe-linked dyes that allow for detection of one or more targets in the same well, passive reference dyes are not influenced by the PCR process and can be used to normalize against several variables that create well-to-well variation.

Common sources of process errors that can compromise your data

Bubbles in wells
Evaporation
Condensation or droplets
Instrument issues, such as electrical surges

Monitoring ROX fluorescence in a Multicomponent Plot enables researchers to detect various quality control issues in a qPCR run. To troubleshoot your data using the Multicomponent Plot, review the shape of the ROX dye signal:

If the ROX dye reading …

Possible issues

Remains flat throughout run

Expected result. ROX fluorescence should remain constant for the duration of the reaction because it is not affected by amplification as shown in the figure below.

Increases during the run

Evaporation may be occurring because the ROX concentration is increasing as the volume decreases.

Momentarily spikes or drops

Sudden changes are interfering with data recording in that well, such as air bubbles or an electrical surge.

Sample multicomponent plot of expected result using ROX dye

Blog Post: The Purpose of ROX reference dye in Real-Time PCR (qPCR)

Instrument and master mix compatibility

ROX dye has been qualified for use on all Applied Biosystems real-time PCR Instruments. ROX dye is detected in Dye Channel 4 (excitation filter: 580 ± 10 nm, emission filter: 623 ± 14 nm).

Dye compatibility with QuantStudio systems

Channel	1	2	3	4	5	6
Excitation filter	470 ± 15 nm	520 ± 10 nm	550 ± 10 nm	580 ± 10 nm	640 ± 10 nm	662 ± 10 nm
Emission filter	520 ± 15 nm	558 ± 12 nm	586 ± 10 nm	623 ± 14 nm	682 ± 14 nm	711 ± 12 nm
Examples of compatible dyes	FAM, SYBR Green I, SYTO 9, fluorescein, PTS dye	VIC, JOT, TET, HEX, Yakima Yellow, SUN	TAMRA, NED, BODIPY TMR-X, ABY, Cy3	ROX, Texas Red, JUN	LIZ, Cy5, Mustang Purple	Cy5.5, Alexa Fluor 680, TYE 685
QuantStudio 1 system	✓	✓		✓
QuantStudio 3 system	✓	✓	✓	✓
QuantStudio 6 Pro system	✓	✓	✓	✓	✓
QuantStudio 5, 7 Pro, and 12K Flex systems	✓	✓	✓	✓	✓	✓

Our standalone ROX kit is compatible with all other TaqMan and TaqPath master mixes and all TaqMan Assays that do not contain JUN, Texas Red, or Alexa 594 as the reporter dye.

ROX can also be used with competitor master mixes if the mix does not contain a coloring agent or passive reference dye. Optimization will also be necessary.

Sample data

How ROX dye normalizes qPCR amplification plots.

(A) Without ROX dye normalization, bubbles in wells affect fluorescence readings. (B) When data was normalized with ROX dye as a passive reference, aberrations caused by the bubbles are removed.

Application Note: ROX passive reference dye for troubleshooting real-time PCR

Ordering information

ROX dye is available as a standalone kit or in a selection of Applied Biosciences qPCR master mixes.

TaqMan and SYBR master mixes that contain ROX premixed:

For Research Use Only. Not for use in diagnostic procedures.

*TaqPath master mixes are general purpose reagents labeled 'For Laboratory Use' and manufactured in an ISO 13485-certified and FDA-registered facility that adheres to cGMP principles.

If the ROX dye reading …	Possible issues
Remains flat throughout run	Expected result. ROX fluorescence should remain constant for the duration of the reaction because it is not affected by amplification as shown in the figure below.
Increases during the run	Evaporation may be occurring because the ROX concentration is increasing as the volume decreases.
Momentarily spikes or drops	Sudden changes are interfering with data recording in that well, such as air bubbles or an electrical surge.