Multiplexing qPCR

Multiplexing Recommendations

Here we offer the navigational tools to select sequences, choose dye-sets, and then order the oligos for successful amplification. Only LGC Biosearch Technologies offers dyes and quenchers that span the spectrum to maximize your multiplexing with 2-plex, 3-plex, 4-plex, and 5-plex qPCR.

Where to begin? Select the right dyes for your instrument.

Spectral Overlay Tool for Multiplexed qPCR

Visualize the absorption and emission spectra for common fluorescent dyes and quenchers. Overlay them as multiplexed sets according to recommended combinations for each qPCR device.

Multiplexing requires oligo sequences that are carefully chosen to promote uniformly high amplification efficiency. This effort is needed to prevent any one assay from dominating since they all compete for the same pool of reaction components. In particular, primer sequences should be avoided that have significant alignment to one another since primer-dimers artifacts can reduce the efficiency and sensitivity of the assay-set.

Such considerations are automatically applied when selecting oligos with RealTimeDesign (RTD), a powerful software program that is available for free through your web browser. RTD designs multiplexed assays all at once to minimize trial and error.

Method

Designing Oligos for Multiplexed Assays is as Easy as 1-2-3

1. Log into RealTimeDesign (RTD) software at www.biosearchtech.com. Simply paste into the entry field all of your target genes that you will detect within a single reaction, either as accession numbers or as raw sequences. Instruct RTD to multiplex the  assays together before commencing design, and proceed to choose a combination of fluorophores.
2. Dyes should be chosen according to the detection capabilities of your real-time PCR thermal cycler. Device Optics considerations are covered in the next section, but our recommended dyes are quickly assigned according to your brand of PCR thermal cycler.
3. RTD will rapidly design your multiplexed assay. You can simply add the oligos to your shopping cart directly, or you may export them from the software. Exported sequences can be pasted into our online ordering form for purchase at a later time.

Figure 1: screenshot from RealTimeDesign showing oligo sequences and their properties. This software is available for free public use on the web at: www.biosearchtech.com

The performance of a 5-plex assay designed using RTD can be viewed in the following poster, A Rapid Bioinformatic Engine for Multiplexed qPCR Design.

Each probe in the multiplexed assay set must have a unique reporter dye in order to separate the amplifications according to fluorescent signal. Every reporter offered by Biosearch can be perfectly partnered with a Black Hole Quencher® dye to achieve fluorogenic probes with excellent signal-to-noise values. BHQ-quenched probes are perfect for multiplexing since they lack the background fluorescence of TAMRA-quenched probes.

Figure 2: the normalized emission spectra for a series of fluorophores provide a reference to choose potential candidates for multiplexing.

When multiplexing, the fluorescent signal from one reporter can bleed into adjacent channels. This crosstalk is avoided by selecting dye-sets with distant emission spectra that are easily resolved.

Figure 3: optimal reporters for a pentaplexed assay on the Rotor-Gene™ Q are identified by comparing spectra to the instrument’s filter specifications.

Optical Specifications are different for each thermal-cycler. When choosing fluors, we consider the excitation source, whether it is LASER, lamp, or LED, as well as the filters for detection.

Figure 4: signal bleeds through from CAL Fluor Red 610 (red traces) into the channel detection Quasar 670 (blue traces). Crosstalk is subsequently removed using software settings.

Crosstalk is fluorescent bleed-through between adjacent channels. If unanticipated, crosstalk can produce false positive amplifications and impair quantification.

After synthesizing the primers and probes, further effort is needed to validate a multiplexed assay. Here we present common guidelines, recognizing that certain applications may require additional steps.

The performance of each assay should remain uncompromised upon combining them together and so cycle threshold values for individual reactions should agree with those multiplexed.  It is common for the magnitude of fluorescent signal to become suppressed upon multiplexing, but not a problem as long as the amplifications overlay during the exponential phase.

Figure 5: multiplexed reactions (black) overlay those amplified individually (green) at a given target quantity.

Figure 6: four assays signaled by different reporters are amplified across a dilution series of their gene targets. The fifth assay signaled by FAM is amplified from a fixed, high copy number in all multiplexed reactions (inset).

Method

Determining Multiplexed Amplification Performance

1. Templates are needed to trigger amplification and qualify each assay’ performance. Templates can be a synthetic amplicon, a linearized plasmid, or even a PCR product from a previous reaction.
2. A dilution series of each template should span a large range of copy number, to determine amplification efficiency and assay sensitivity. Each assay should be qualified individually and in combination.
3. At any given template quantity, the cycle threshold values for individual amplifications should overlay those multiplexed.
4. In certain applications such as gene expression analysis, one or more targets may be in vast excess over others. We recommend qualifying assay performance by amplifying from disproportionate copy number.
5. After the amplification performance has been validated, you are ready to quantify your unknown samples. Quantification is only reliable should these samples amplify with the bounds of the validation (e.g. within the range of the dilution series).
6. Assays that suffer upon multiplexing can be improved with a master mix intended for multiplexing that has been supplemented with additional components. More information on master mix formulation can be found in the following reference:
   Four-color multiplex reverse transcription polymerase chain reaction--overcoming its limitations. Persson, K.; Hamby, K.; Ugozzoli, L.A. Anal. Biochem. 2005 Sep 1;344(1):33-42.

You are now ready to flex your multiplex power! 

Additional information on the intricacies of PCR multiplexing can be found in the following poster: Intricacies of PCR Multiplexing As Revealed Through a Pathogen Detection Assay (PDF 1.7 MB)

A variety of purposes are served by combining several assays together into one. Here are a few key examples from the literature.

Internal Positive Control

An internal positive control is a secondary assay that amplifies with every experiment, to confirm that genetic material is properly extracted and that cDNA is efficiently generated. Representing either an endogenous target such as a native gene or an exogenous template spiked into the sample, this control can be assayed in separate wells on the PCR plate but provides even more assurance if combined into the same wells for detecting your gene of interest. Only by multiplexing together with your other assays can you have complete confidence in every single reaction on your plate.

Development of an Internal Positive Control for Rapid Diagnosis of Avian Influenza Virus Infections by Real-Time Reverse Transcription-PCR with Lyophilized Reagents. Das, A.; Spackman, E.; Senne, D.; Pedersen, J.;Suarez, D.L. J Clin Microbiol. 2006; 44(9): 3065–3073.

Replicate Assays

Since the read-out from qPCR assays can occasionally mislead, some researchers detect the same gene with several distinct assays. This redundancy boosts confidence in quantification since several factors produce contradictory results: degradation of the RNA transcript, differing efficiencies during reverse transcription, and nonspecific amplification from off-target regions. Reagent handling is then streamlined by combining together these replicate assays into a single reaction, making more efficient use of pipetting steps, PCR reagents, and sample material.

Quantification of mRNA using real-time RT-PCR. Nolan, T.; Hands, R.E.; Bustin, S.A. Nat Protoc. 2006;1(3):1559-82.

Distinguishing Strains

Related organisms may share many similar sequences making them difficult to distinguish them from their nearest neighbors. For example, strains of microbes harboring certain plasmids demonstrate a virulence absent from relatives that are otherwise identical. Researchers amplify multiple signatures to properly identify these strains and diagnose their threat to health. If this molecular diagnostic becomes a routine test then multiplexing can facilitate commercialization so that fewer reactions are required to assign each result.

Multiplexed detection of anthrax-related toxin genes. Moser, M.J,; Christensen, D.R.; Norwood, D.; Prudent, J.R. J Mol Diagn. 2006 Feb;8(1):89-96.

Want to learn more about Multiplexed qPCR?

Contact our Technical Support Team at techsupport@biosearchtech.com.

Multiplexed qPCR combines several PCR assays together into one, to conserve sample material and avoid well-to-well variation. The targets are amplified simultaneously but detected independently using reporters with distinct spectra. Individual signals are recorded and resolved into fluorescent intensities that correlate with each amplification.

Multiplexed PCR requires more time to implement than the same assays run independently, but the rewards justify the investment. A single reaction yields additional data and confidence in the results, and cost savings to an optimized assay run repeatedly -- ideal for commercial products and high-throughput processes.