Influence of ROX on the Ct value

February 1, 2019 Science

Influence of ROX on the Ct value

Why is ROX used as a passive reference dye in real-time PCR and what influence does ROX have on the evaluation of PCR in real-time PCR? Can too much ROX negatively affect the evaluation?

Ct value, optical systems, and passive reference dye

A Ct value (also Cp or Cq value, abbreviations for threshold cycle) is a theoretical value derived from the intersection of a pre-set lower value (threshold) and a given amplification curve. It indicates the relative concentration of the initially present target DNA in the qPCR reaction.

Various sample-independent factors have a more or less significant influence on the Ct value. These include the optical system, the PCR master mix, the setting of the threshold value, and the setting of the baseline value. Additionally, whether a passive reference dye like ROX is required for a specific device or is already present in the qPCR reaction also plays a role.

How does a passive reference dye affect the Ct value?

It is important to understand how real-time PCR devices work. In many devices, especially older types, there is a stationary light source that illuminates all wells of the PCR block. Simultaneously, the detection system is also stationary. This results in different light paths to the various wells and back to the detection source. The variation in light path lengths leads to different absolute fluorescence measurements for each well, even if the same amount of target DNA is present.

For example, wells with a shorter light path (typically located in the middle of the block) have a higher fluorescence value compared to a well with a longer light path (typically on the edge of the PCR block). The differences in light paths and thus the absolute fluorescence measurements would influence the result of the qPCR evaluation if the measured fluorescence values were not normalized.

Normalization of qPCR measurements using a reference dye

During qPCR data processing, the sample fluorescence for each well is first subtracted from the value of the reference dye. To normalize the fluorescence intensity, the qPCR software divides the emission intensity of the reporter dyes by the emission intensity of a reference dye.

ROX (Carboxy-X-Rhodamine) has become commonly used as a passive reference dye in real-time PCR systems to normalize differences in fluorescence values. ROX is not involved in the PCR reaction and does not interfere with it at "normal" concentrations of 0 nM to 600 nM (no interaction with DNA or DNA polymerase), so adding ROX to a qPCR master mix results in a constant fluorescence signal throughout the amplification. The constancy of ROX fluorescence can therefore be used as a baseline for normalization.

The available qPCR master mixes are now mainly offered with different ROX concentrations because devices from different manufacturers have different optical configurations that require different ROX concentrations as a baseline for normalization. This is primarily due to the type of excitation source (laser, LED, etc.), the optics used, and the method by which the excitation light is directed to the well (fixed light source, moving sled, fixed source for each well).

To account for these differences, Genaxxon offers three different concentrations of ROX: without ROX (M3023, M3045), low ROX (M3011, M3031), high ROX (M3010, M3052). However, customer experiments show that even devices that require 50 nM ROX can work with a master mix containing 500 nM ROX. This may require a change in the settings on the qPCR device.

Normalization calculation - normalized reporter fluorescence intensity Rn

To normalize qPCR data, the fluorescence emission intensity of the reporter dye (for example, SYBR® Green) is divided by the fluorescence emission intensity of the passive reference dye. This ratio is called normalized reporter intensity or Rn (see Table 1). To further improve the measurements, the so-called Rn+ and Rn- are needed. Rn+ is the Rn from PCR reactions that give a signal above the threshold. Rn- is the baseline fluorescence that can be derived from the early cycles or set as the threshold. To determine the reporter fluorescence intensity above the baseline, ΔRn is calculated as (Rn+) - (Rn-).

Table 1: Rn calculation for two differently positioned wells (centrally or peripherally located). The change in fluorescence is corrected by normalizing the fluorescence reporter signal to the fluorescence signal of the passive reference dye.

Fluorescence intensity, relative fluorescence units (RFU)

Well	Reporter dye	Passive reference dye	Rn
Center of block	3000	1500	2
Periphery of block	2000	1000	2

Further reading: Ruijter, J. M., Zhao, S., Spiess, A. N., Boggy, G., Blom, J., Rutledge, R. G., … Vandesompele, J. (2013). Evaluation of qPCR curve analysis methods for reliable biomarker discovery: Bias, resolution, precision, and implications. Methods, 59(1), 32–46. https://doi.org/10.1016/J.YMETH.2012.08.011