Will I see a difference in my qPCR data

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Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye


Question:

Will I see a difference in my qPCR data if I change from an older real-time PCR reagent that contained a high or low ROX concentration to a Bio-Rad supermix containing a universal reference dye?


Answer:

Users of Bio-Rad real-time PCR detection systems will not see any significant differences in their qPCR data because the new supermix formulations simply incorporate a universal passive reference dye, which is ignored by our optical detection systems. Users of ROX-dependent real-time PCR systems (for example, Life Technologies' systems) will now be able to use any of our universal real-time PCR reagents without having to determine which product to use.

If you were using an older product with ROX, you may see a slight shift in Cq values of less than ±0.5. However, since all the Cq values are shifted by the same amount, the relative quantitation data remain the same, with no impact on the final results.

Comparison of Universal Reference Dye Performance on a Life Technologies AB7900 using an ACTB cDNA assay

Comparison of universal reference dye performance on a Life Technologies AB7900 system using an ACTB cDNA assay. Amplification traces for iTaq™ SYBR® Green supermix with ROX () and iTaq™ universal SYBR® Green supermix with the new universal reference dye (). An earlier Cq shift of <0.3 is observed with the new universal dye.

See Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye in our Applications and Technologies pages to learn why passive reference dyes are required for data normalization in some real-time PCR systems and not others. Although Bio-Rad real-time PCR detection systems do not require ROX for internal normalization, Bio-Rad’s universal real-time PCR supermixes are designed for high performance on all real-time PCR detection systems, both systems requiring ROX normalization and ROX-independent systems.

View our Understanding Real-Time Supermixes tutorial to learn more about qPCR data analysis and how to compare supermix performance. 


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