Do the levels of relative fluorescence units (RFUs) for a qPCR reaction have an impact on my data?

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


Question:

Do the levels of relative fluorescence units (RFUs) for a qPCR reaction have an impact on my data?


Answer:

No. The Cq values will remain the same regardless of the RFU level. The reporter dye is simply used to yield a signal in the exponential phase of PCR so that a Cq value can be derived. The absolute magnitude of the fluorescence level is thus unimportant.

The RFU signal is determined by several factors including

  • The reporter dye used — green dyes such as FAM and SYBR® are brighter than Cy5 and other dyes closer to the red end of the spectrum
  • Reagent formulation — components such as buffers and salts affect pH and ionic strength, which in turn affect the RFU; thus, RFU levels may vary among real-time PCR supermixes from different vendors

Comparison of Universal Reference Dye Performance on a Life Technologies AB7900 using an MMP9 cDNA assay

Comparison of universal reference dye performance on a Life Technologies AB7900 system using an MMP9 cDNA assay. Amplification traces for iTaq™ SYBR® Green supermix with ROX () and iTaq™ universal SYBR® Green supermix with the new universal reference dye (). No Cq shift was observed in the data.

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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