An integral requirement of the reformulation was to ensure that no significant changes were noted in performance. The Bio-Rad R&D team validated these updated supermixes with an extensive set of qPCR assays to assess the following attributes:
- Consistent assay results for relative expression levels
- PCR efficiency
- Dynamic range
The same validation process was used in the reformulation of SsoFast™ probes supermix to create SsoAdvanced universal probes supermix.
In terms of absolute Cq values, although the vast majority of data were identical to those obtained with the previous formulation, we noted a few assays that demonstrated Cq shifts of ±0.5. This shift does not impact qPCR data in any manner, as Cq values in and of themselves are meaningless values.
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.