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qPCR Probes—selecting the best reporter dye and quencher

You may be new to qPCR and are uncertain about which probe dyes are compatible with the qPCR instrument in your laboratory; or maybe you are designing probes for your assays, and have questions about quencher options. Here we provide some advice on how to select appropriate dyes and quenchers for your probe-based qPCR assays.

Reporter dyes

Instrument compatibility. Selecting an appropriate reporter dye for qPCR analysis will depend on the type of instrument you are using and the compatibility of the dye with the instrument. Refer to your instrument manufacturer’s guidelines for information specific to your particular instrument. Figure 1 provides a list of reporter dyes compatible with common instrumentation. FAM is the most popular of these dyes, and often provides greater assay sensitivity than some of the other dyes available.

Figure 1. Instrument compatibility with reporter dyes. Don’t see the instrument or dye you are looking for? Contact applicationsupport@idtdna.com.

Multiplexing. For multiplexing applications, each target must be identified by a separate reporter dye. Select reporter dyes with the least amount of spectral overlap. Figure 2 provides the emission wavelengths for a variety of popular dyes. It also indicates the recommended quenchers that can be used with those dyes. As a general rule, select a dye with a strong signal, such as FAM, for any low copy transcripts. Lower signal fluorophores can then be used for the more abundant transcripts.

Figure 2. Wavelengths of select IDT dyes, and the appropriate quenchers. This diagram can help with your selection of the appropriate quencher for your fluorophore. IDT offers the ZEN™/Iowa Black™ FQ and Iowa Black® RQ quencher options for qPCR probes, and each of these provides efficient quenching for a different range of dyes. For clarification on these options, please contact our Scientific Applications Support team at applicationsupport@idtdna.com.

Quenchers

Traditional dark quenchers absorb broadly and do not emit light, which allows use of multiple reporter dyes with the same quencher (Figure 2). This characteristic allows for expanded options for multiplex assays. Dark quenchers reduce signal cross-talk, simplifying reporter dye detection, making them compatible with a broad range of image analysis instruments. Examples of dark quenchers include Black Hole Quenchers, and Iowa Black® FQ and RQ.

IDT has developed the internal ZEN™ Quencher, also a dark quencher. It can be used in addition to the 3’ quencher Iowa Black FQ, resulting in double-quenched probes (5’FAM/ZEN/3’IBFQ; Figure 3A). These double-quenched probes generate less background and increased signal compared to probes containing a single quencher (Figure 3B). See the blue Product focus box below for more information.

A. Positioning of ZEN™ Internal Quencher within double-quenched probe.

B. ZEN™ Double-Quenched Probes increase detection sensitivity compared to single-quenched probes.

Figure 3. ZEN™ Double-Quenched Probes produce low background and high signal intensity in qPCR experiments. (A) Positioning of the ZEN Quencher in a Double-Quenched Probe (5’FAM/ZEN/3’IBFQ). (B) qPCR assays that use the same primer and probe sequences targeting the ACTB locus, but use 5’ FAM dyes with 5 different quenchers, were compared. All assays were run in triplicate with 0.5 ng cDNA and Applied Biosystems TaqMan® Gene Expression Master Mix under standard cycling conditions on the Applied Biosystems 7900HT. Key: ZEN/IBFQ = ZEN™–Iowa Black® FQ, BHQ = Black Hole Quencher®, and IBFQ = Iowa Black FQ quenchers.

IDT supplies commonly used dark quenchers as well as the proprietary dark quenchers, Iowa Black FQ, Iowa Black RQ, and the internal ZEN Quencher. TAMRA is also a quencher option for a FAM reporter dye.

More questions? We can help.  Email our experts for assistance with your qPCR project.

Author: Ellen Prediger, PhD, is a Senior Writer at IDT.

Product focus: ZEN™ Internal Quencher

The ZEN Internal Quencher enables the production of Double-Quenched Probes that generate less background and increased signal.

Double-Quenched Probes that include the ZEN Internal Quencher are available with 5′ FAM, TET™, HEX™, MAX™, or JOE fluorophores, and a 3’ IBFQ quencher.

ZEN Double-Quenched Probes display consistently earlier Cq values and improved precision when compared to traditional probes with only a 5′ fluorophore and 3′ quencher. 

To learn more, download the ZEN Double-Quenched Probe Overview.

Additional reading

Recommended Dye Combinations for Multiplex qPCR—Recommendations for selecting dyes for multiplex qPCR that minimize background and avoid overlap of fluorescent signals. Included is a table of compatible dyes for multiplexing on common qPCR instruments and a list of suggested quenchers.

Designing PCR Primers and Probes—General guidelines for designing primers and probes and for choosing target locations for PCR amplification

Two Quenchers Are Better Than One!—Including a second, internal quencher in qPCR probes shortens the distance between 5’ dye and quencher and, in concert with the 3’ quencher, provides greater overall dye quenching, lowering background and increasing signal detection in qPCR experiments.

PrimeTime® qPCR Application Guide—This 93 page booklet provides guidance on the entire qPCR process, from RNA isolation to data analysis and troubleshooting.

© 2015 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.






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