Multiplexing options for probe-based qPCR with unique red qPCR dye
Biotium, a life science reagent supplier and inventor of the widely used EvaGreen qPCR dye, has released EvaRuby dye for qPCR and high-resolution melt (HRM) analysis.
This spectrally unique red fluorescent intercalating dye can be incorporated into probe-based qPCR assays, expanding multiplexing options for probe-based qPCR. EvaRuby dye is compatible with ~470 nm excitation and ~610 nm emission filters.
While various DNA binding dyes with different fluorescence emission properties have been published for use in qPCR and HRM, only the green fluorescent dyes SYBR Green I, SYTO 9 and EvaGreen dye have widespread use in these applications. EvaGreen dye is currently the only dye suitable for qPCR and HRM in the same reaction; however, due to its relatively broad emission, EvaGreen cannot be combined with red PCR probes for multiplexing or HRM in probe qPCR reactions.
As a solution, Biotium scientists have developed EvaRuby, a unique dye with a much longer Stokes shift of 480/613 nm (excitation/emission). This allows excitation of EvaRuby dye by the same green/blue channel as EvaGreen dye, and detection in the orange (JUN or ROX) channel. As a result, EvaRuby dye has minimal cross-talk in standard probe detection channels and enables the combination of dye-based and probe-based qPCR in a single reaction. In addition, EvaRuby dDye is validated for HRM analysis and may be used as an internal control for probe-based qPCR to detect non-specific amplification that goes undetected by target-specific probes. EvaRuby dye is also suitable for singleplex qPCR, LAMP and gel electrophoresis.
EvaRuby dye has been validated on Applied Biosystems QuantStudio 5 96-well and Qiagen Rotor-Gene Q instruments, with potential compatibility for other qPCR instruments capable of acquiring data using ~470 nm excitation and ~610 nm emission filters.