This white paper, authored by scientists at Promega Corporation, explores the optimization of passive reference dye usage with the PowerQuant® System on the QuantStudio™ 5 Real-Time PCR System. The PowerQuant® System is widely used in forensic DNA quantification, offering sensitive detection of total human and male DNA, as well as data to assess DNA degradation and amplification inhibition. Developmental validation of the PowerQuant® System was performed on the Applied Biosystems™ 7500 Real-Time PCR System with subsequent evaluation on the QuantStudio™ 5 platform.

As laboratories began using the QuantStudio™ 5 platform, some observed anomalous results, such as standard curve slopes outside recommended ranges and occasional low-level male DNA signals in female-only samples. These findings prompted further evaluation to better understand platform-specific performance characteristics.

Through a series of experiments, Promega examined these observations and identified spectral crosstalk between the IPC dye and the passive reference dye (CXR) on the QuantStudio™ 5 instrument as a primary factor.

The team evaluated a modified protocol incorporating an additional 150 nM of the CXR passive reference dye in the qPCR reaction. This modification resulted in standard curve slopes within the recommended range and eliminated spurious low-level male signals in female-only samples. Reassessment of analysis thresholds with the increased CXR concentration I demonstrated sensitivity comparable to the standard PowerQuant protocol.

This white paper serves as guidance for laboratories using the PowerQuant® System on the QuantStudio™ 5 Real-Time PCR System that observe these behaviors. Laboratories may use the information presented to inform their own instrument-specific evaluation.