1. Instrumentation and Dye Selection
This one is pretty easy. For a successful multiplex, your instrument needs to be able to excite each of your reporter dyes. Although there are dyes on the market that excite in the far red, you'll be safe using an instrument which excites over the visible spectrum (350-750nm). Biosearch Technologies has developed two essential tools to assist you with dye selection:
- an interactive spectral overlay chart that allows you to compare spectra and pair dyes with BHQ® dyes (see below to access link)
- a chart displaying multiplexing recommendations for your real-time pcr instrument (see below to access link)
2. Probe and Primer Design
Biosearch provides a free design program called RealTimeDesign™ software which takes the struggle out of probe and primer design. For a successful qPCR multiplex experiment, primers and probes must be designed to avoid secondary structures and cross hybridization. Dyes should be chosen with distinct emission and excitation spectra in order to minimize crosstalk. RealTimeDesign software designs the sequences for your primers and probes, and selects appropriate reporter dyes and quenchers for your instrument, taking the guesswork out of multiplex design.
3. Dealing with Crosstalk
Crosstalk is fluorescent signal bleed-through between channels. Although unavoidable, crosstalk can be controlled by calibrating your instrument before running the experiment. Biosearch offers calibration standards, SuperRoxTM dye and instructions on their usage. The figure to the right is showing signal bleeds from CAL Fluor® Red 610 dye (red traces) into the channel detection Quasar® 670 dye (blue traces). Crosstalk is subsequently removed using software settings.
That's pretty much it. To help give you a quick jump start in developing a multiplexed qPCR assay, we are offering a ValuProbeTM BHQ Probe promotion where you will have a wider selection of fluorophores. For only $95 and for a limited time only, you can choose from FAM, CAL Fluor Orange 560, CAL Fluor Red 610, and Quasar 670 dyes. Click here to learn more details about our ValuProbe promotion.
Still new to the concept of multiplexing qPCR? Request a Multiplex Brochure from Biosearch Technologies, today!