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.
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. Table 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.
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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.
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 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 2).
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.
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.
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