TECHNICAL NOTE

Guidelines for 3-color multiplex assay design for

optimized performance with Crystal Digital PCR™
By measuring several targets in a single reaction, multiplex assays enable users to conserve precious sample volume and to save time, re-
agents and costs. In addition, as all targets are amplified and measured in the same reaction, multiplexing improves quantification precision
by reducing pipetting errors that contribute to sample and reagent variations when performing separate single-plex reactions. Multiplexing
in Crystal Digital PCR™ is as sensitive and accurate as single-plexing. Careful assay design and assay optimization steps help realize the
more complex reaction composition, containing a high number of primers and probes for the amplification of several DNA targets in a single
PCR reaction. The Crystal Miner software is an open data analysis software that provides intuitive tools to help optimize and troubleshoot
multiplex assays. This Technical Note provides straightforward guidelines to facilitate multiplex assay design using the naica® system.

Guidelines for experimentally evaluating primer

and probe performance
y Stilla ® recommends using the naica ® multiplex PCR MIX,
which is specially formulated for optimal multiplexed Crystal
Digital PCR™ performance.
y It is imperative to start with a Crystal Digital PCR™ run of
the individual reaction composition in the single plex format
first. Each primer/probe/target requires a performance
verification in the less complex reaction mix before
proceeding to multiplexing. For example, for a 3-plex
assay, three individual single-plex reactions should first
be performed on control nucleic acid target templates
before combining the reagents in a multiplex reaction.
When performing a single-plex reaction, a single positive
population is expected.
y For optimized multiplex assay performance, it is important
to consider the final sample matrix and composition (e.g.,
short fragmented DNA for assay design targeting circulating
cell-free DNA, more intact DNA fragments for assay design
targeting genomic DNA).
y The DNA template used should be devoid of contaminants
and potential inhibitors. Synthetic oligos can be used as
templates for assay optimization if final sample material is
rare or not readily available.
y A range of elongation temperatures should be evaluated for
each single-plex reaction to determine the optimal reaction
temperature at which there is good separability between pos-
itive and negative populations, without nonspecific amplifica-
tion (Figure 1). The Stilla® separability score provided by Crys-
tal Miner software (Figure 2) should be used as a metric to
determine the optimal elongation temperature common to all
probes. If the single-plex reaction is not well optimized, a sec-
ond distinct amplified population may be apparent due to, for
example, undesired probe interactions. In addition, non-spe-
cific amplification can result from one of several unoptimized
parameters, including primer/probe dimers or primer/probe
non-specificity. In this case, various methods can be em-
ployed to limit the amplification of non-specific sequences,
such as increasing the annealing temperature, performing a
* https://www.gene-pi.com/item/digital-pcr-assay-optimization-2/ touchdown PCR or redesigning primer sequences. It is im-
https://www.gene-pi.com/item/primers-and-probes-2/ portant to evaluate the primer and probe interactions using
adapted in silico tools before testing the reagents in vitro.
Continued

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 Figure 2 | Separability score is based on the distance between clusters,
and positive and negative cluster spreads. The Separability score is
automatically computed by the Crystal Miner software and can be found
under the Advanced QC tab.

Figure 1 | Crystal Miner software 1D-dotplots of single-plex reactions

showing the fluorescence intensities obtained in the blue, green and
red detection channels across a range of elongation temperatures from
60 °C to 65 °C. The black box indicates the shared optimal elongation
temperature selected for the single-plex reactions. Separability scores
(*) can be used to determine the optimal elongation temperature for
amplification of the 3 targets.

y Perform multiplex Crystal Digital PCR™ with all primers and

probes at the selected elongation temperature and evaluate
the reaction performance using the separability score as a
guide. If necessary:
y Adjust the number of PCR cycles — It is recommended
to start with 45 cycles and to increase the cycle numbers
for further optimization of the separability between the
positive and negative populations.
y Adjust primer and probe concentrations — for the
naica ® system, the recommended primer and probe
concentration range from 0.125 to 1 μM (Figure 3). For
multiplex assay design it is recommended to start at
the low end of the concentration range to minimize
the complexity of the reaction and reduce the volume
occupied by primers and probes.
y Use modified bases such as locked nucleic acid (LNA)1 Figure 3 | Crystal Miner software 1D-dotplots showing the
bases or a minor groove binder (MGB)2 to increase the fluorescence intensities obtained in the blue detection channel across
a range of increasing primer (top panel) and probe (bottom panel)
Melting temperature (Tm) of the probe while keeping a concentrations. The black boxes indicate the selected concentrations
short length (<20nt if possible). However, no more than to be carried over for use in the multiplex assay based on favorable
2 MGBs are recommended in a multiplex assay due to a separability scores and low primer and probe concentrations.
risk of decreased amplification (*: separability score)
Continued

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Figure 4 | Examples of interactions between primers and probes. A) Interaction between the probe of target 1 and the reverse primer of target 2 (R2 target
2, red box). This interaction is not detected when using the reverse primer R1 target 2. In this example, R1 target 2 should be selected for the design of the
multiplex assay. B) Interaction between the probe of target 1 and the forward primer of target 2 (F2 target 2, blue box). This interaction is not detected when
using forward primer F1 target 2. In this example, F1 target 2 should be selected for the design of the multiplex assay.

y Evaluate primer and probe interactions — The probability

of homo/hetero dimer formation between primers and/
Technical Note Highlights
or probes used in the same multiplex experiment should y The naica ® system offers flexible 3-color multiplexing
be kept to a minimum. Dimerization can be evaluated, and capabilities.
interaction scores determined with several in silico design
y The Crystal Miner software facilitates multiplex assay
tools (e.g., IDT OligoAnalyzer™ Tool, Primer3, Applied
optimization and troubleshooting.
Biosystems™ Primer Express ® , PREMIER Biosoft Beacon
Designer™) (Figure 4). High concentrations of primers and y The Technical Note provides cost- and time-saving tips to
probes can increase the probability of undesired interactions. design and validate Crystal Digital PCR™ multiplex assays on
Thus, when multiplexing, it is recommended to start with low the naica ® system.
concentrations of primers for all assays (e.g. 0.25 μM), and
y Designing multiplex assays is not complex if the guidelines
increase the concentrations gradually up to 1 μM if needed
provided in this Technical Note are followed.
(for example, to increase the amplification efficiency).
y For a multiplex assay, it is important to compensate for Endnotes
fluorescence spillover to ensure robust quantification.
Using monocolor controls, the Crystal Miner software 1
Locked nucleic acids in PCR primers increase sensitivity and per-
allows to create a compensation matrix adapted formance. Ballantyne KN, van Oorschot RA, Mitchell RJ. Genom-
to a given multiplex panel. For further detailed ics. 2008 Mar;91(3):301-5. doi: 10.106/j.ygeno.2007.10.016 [PMID:
description of fluorescence spillover, please visit 18164179]
https://www.gene-pi.com/item/spill-over-2/. Instructions
for performing fluorescence spillover compensation can 2
3'-Minor groove binder-DNA probes increase sequence specifity at
also be found in the Crystal Miner software User Manual. PCR extension temperatures. Kutyavin IV, et al. Nucleic Acids Re-
search. 2000 Jan;28(2): 655-661. doi.org/10.1093/nar/28.2.655.

To learn more about digital PCR, please visit

Stilla Technologies’ Learning Center at
stillatechnologies.com/digital-pcr
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