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.

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 1 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.

Quenchers

Traditional dark quenchers absorb broadly and do not emit light, which allows use of multiple reporter dyes with the same quencher (Figure 1). 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 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 2). Another quencher is Black Hole Quencher^®-1 (BHQ), BHQ-2, and BHQ-3 (LGC Biosearch Technologies). 

Figure 2. Positioning of ZEN Internal Quencher within double-quenched probe.

These double-quenched probes generate less background and increased signal compared to probes containing a single quencher (Figure 3). See the Product focus box (right) for more information.

Figure 3. ZEN Double-Quenched Probes produce low background and high signal intensity in qPCR experiments. 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. Contact applicationsupport@idtdna.com for assistance with your qPCR project.