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EvaRuby™ Dye, 20X in Water

A spectrally unique visible red fluorescent dye for qPCR, HRM®, and LAMP. Can be combined with commonly used fluorescent qPCR probes with little cross-talk in other channels.

Product Attributes

Detection method/readout

PCR/qPCR, HRM®

Excitation/Emission

480/613 nm (with DNA)

Storage Conditions

Store at -10 to -35 °C

Note: Due to operating restrictions related to our COVID-19 response, primary antibody conjugates of R-PE, APC, PerCP, HRP, or AP are temporarily unavailable. All other antibody options are still available. We apologize for the inconvenience. Please see our easy-to-use Mix-n-Stain™ Antibody Labeling Kits for labeling antibodies with R-PE, APC, tandem dyes, and enzymes.
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500 uL
2 x 1 mL
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Product Description

EvaRuby™ is the first visible red DNA-binding dye for both qPCR and HRM. The dye has unique spectral properties that minimize cross-talk between channels. EvaRuby™ can be used as a typical intercalating dye for qPCR, HRM®, and LAMP. The dye also can be incorporated into probe-based qPCR assays to monitor the reaction in real-time and perform post-qPCR high-resolution melt analysis.

  • Visible red qPCR dye with minimal cross-talk in neighboring detection channels
  • Unique spectral properties: Ex/Em 480/613 nm (with DNA)
  • Combine probe-based qPCR with HRM® in the same reaction
  • Use as an internal control to monitor probe-based qPCR reactions in real-time
  • Avoid wasting time and resources on post-qPCR gel electrophoresis
  • Detect with green ~488 nm excitation and orange ~610 nm emission filters

The unique properties of EvaRuby™ Dye

EvaRuby™ is a visible red fluorescent nucleic acid dye designed for qPCR, as such it is essentially non-fluorescent by itself, but becomes fluorescent upon binding to dsDNA. Unlike EvaGreen® or SYBR® intercalating qPCR dyes, EvaRuby™ has a long Stokes shift and orange fluorescent emission. With excitation/emission peaks at 480/613 nm, EvaRuby™ Dye is excited using the same green/blue channel as EvaGreen® but is detected in the orange (JUN™ or ROX) channel. As a result, EvaRuby™ has minimal cross-talk in other detection channels and therefore can be incorporated into singleplex and multiplex probe-based qPCR assays (Figure 1).

Figure 1. The effect of 1X EvaRuby™ Dye on the amplification and detection of the TFCR gene by a CAL Fluor® Red 610 hydrolysis probe in 10-fold dilutions of Human gDNA. Both EvaRuby™ and the CAL Fluor® Red 610 probe share the 610 nm emission channel. However, since EvaRuby™ is minimally excited by the Qiagen Rotor-Gene® Q Orange (585 nm) excitation channel used for the probe, the standard curve generated by the probe is practically unchanged by the presence of EvaRuby™.

While EvaRuby™ can be used as a traditional intercalating qPCR dye to perform real-time qPCR, melt curve analysis, LAMP, and HRM®, the dye’s main advantage is that it can be incorporated into probe-based qPCR assays.  Unlike other intercalating dyes, EvaRuby’s™ unique spectral properties allow it to be combined with commonly used fluorescent qPCR probes with little cross-talk in other fluorescence channels. The dye can be added to the reaction mix during reaction setup without affecting probe specificity (Figures 1, 2) while expanding the capabilities of probe-based qPCR to include real-time troubleshooting and post-qPCR high-resolution melt analysis (Figure 3). Note that for traditional single-color dye-based qPCR, we still recommend using EvaGreen® Dye.

Figure 2. The effect of EvaRuby™ on Ct determination by hydrolysis probes. Three independent qPCR assays were performed for each probe dye using common probe-based qPCR settings. Each point is the Ct value determined by the indicated probe. Despite some overlap in the excitation or emission spectra, the Ct values obtained by the probes in a standard probe-based assay without EvaRuby™ (rings) are minimally different from the Ct values obtained when probes are used in combination with EvaRuby™ (circles).
Figure 3. High Resolution Melt Data Difference Plot. EvaRuby™ Dye at 1X in Forget-Me-Not™ Universal Probe qPCR Master Mix (Cat. No. 31043) was used to detect the Applied Biosystems® MeltDoctor™ HRM Positive Control Kit. Data was acquired using the Qiagen® Rotor-Gene® Q, 0.1°C/2 sec, with a 470 nm excitation and 610 nm emission filter. Data analysis was performed with uAnalyzeSM (https://dna-utah.org) using baseline subtraction. EvaRuby™ Dye can clearly distinguish each homozygous variant as well as the heterozygous variant.

A New Internal Control for Probe-based qPCR

Dye-based qPCR has low target specificity but has the advantage of performing a rapid and facile confirmation of results with melt curve analysis. On the other hand, probe-based qPCR benefits from high target specificity but is not compatible with melt curve analysis. Instead, confirmation of the qPCR results is often done by gel electrophoresis, a labor-intensive step that increases the time to results. Using EvaRuby™ in combination with probe-based qPCR enables the use of probe for high target specificity together with an intercalating dye for melt analysis and confirmation of results. Furthermore, because EvaRuby™ signal is independent from probe signal, it acts as an internal control for PCR amplification. For example, despite successful amplification, a degraded or suboptimal probe may fail to bind to the amplified target sequence and as a result, qPCR would incorrectly report amplification failure. However, with EvaRuby™ present, amplification would still be detected during qPCR and can be identified with melt curve analysis.

Instrument Compatibility

EvaRuby™ requires detection using a custom qPCR detection channel. Your qPCR instrument must have ~470 nm excitation and ~610 nm emission filters, as well as the option to acquire data using this non-standard filter combination. Instruments with paired excitation/emission filters may not be compatible with the dye. EvaRuby™ has been validated on Applied Biosystems® QuantStudio™ 5 96-well and Qiagen® Rotor-Gene® Q instruments. A compatibility table for other instruments is available in the PI; however it is important to confirm compatibility by checking your instrument technical specifications or by contacting the instrument manufacturer before purchasing EvaRuby™ Dye.

If your qPCR instrument requires a passive reference dye, use VeriFluor™ Far-Red (Cat. no. 29087) for optimal results. VeriFluor™ Far-Red is detected in the red (Cy®5 or Mustang Purple™) fluorescence channel. High ROX concentrations (~500 nM) are incompatible with EvaRuby™. Using low ROX concentrations (~50 nM) for normalization is suboptimal and will result in a small increase of the background fluorescence in the EvaRuby™ channel.

 

EvaGreen Dye and applications are covered under granted and pending US and international patents. Rotor-Gene is a registered trademark of Qiagen. QuantStudio, JUN, and Mustang Purple are trademarks or registered trademarks of Thermo Fisher Scientific. Cy Dye is a registered trademark of GE Healthcare. CAL Fluor is a registered trademark of Biosearch Technologies, Inc. HRM is a registered trademark of Idaho Technologies, Inc./BioFire Defense, LLC and its use may require a license.
 

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