A Quick Jump Start to Multiplexing qPCR

Posted on Wed, Jan 06, 2010 @ 10:01 AM

multiplexing qPCRBy now, you've no doubt read about the reductions in error, cost and time that can be achieved by multiplexing real time PCR. A common complaint about multiplexing is that, despite its benefits, it is sometimes considered too much of a hassle. This post will address some key factors to consider when planning a multiplexed qPCR assay.

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)

RealTimeDesign Software2. 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.

Crosstalk Amplifications3. 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!

Useful Tools and Resources:
Multiplex Guide
Interactive Spectral Overlay Tool
Multiplexing Recommendations Chart
RealTimeDesign Software
Instructions for Calibration Standards

Related Products:
ValuProbe BHQ Probes
Dual-Labeled BHQ Probes
Calibration and Reference Dyes

Topics: Real-time PCR, Design Services

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About Biosearch Technologies

As the inventor of the BHQ dyes, Biosearch Technologies synthesizes sophisticated oligos for real time qPCR, molecular diagnostics, and more!  Visit our home page to view our products and services.

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