Multiplex end-point PCR is a powerful tool for genotyping and many other applications. QIAGEN’s multiplex PCR chemistry is optimized for reliable amplification of many different templates with high variability in copy numbers. Thus it enables very quick establishment of a new lab routine and instant success for your multiplex PCR strategy. There is a set of critical factors which we recommend to be regarded for planning and performing this kind of PCR. These will be discussed in detail in the webinar. Additionally, our multiplex PCR chemistry has recently been gaining increasing popularity among scientists who are utilizing it for their next-generation sequencing workflows.

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Critical Factors for Successful Real-Time PCR: Multiplex PCR
Laura Alina Mohr, MSc, Global Market Manager Assoc.
Cover Page 2
1Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Maximizing Real-Time PCR Results: Sample to Insight
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Two-part webinar series
Part 1: Practical Hints and New Solutions for Successful Real-
Time PCR Studies
Part 2: Critical Factors for Successful Real-Time PCR, Multiplex
PCR
Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

3. Sample to Insight
Maximizing Real-Time PCR Results: Sample to Insight
3
Two-part webinar series
Part 1: Practical Hints and New Solutions for Successful Real-
Time PCR Studies
Part 2: Critical Factors for Successful Real-Time PCR, Multiplex
PCR
Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Legal disclaimer
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QIAGEN products shown here are intended for molecular biology
applications. These products are not intended for the diagnosis, prevention, or
treatment of a disease.
For up-to-date licensing information and product-specific disclaimers, see the
respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and
user manuals are available at www.QIAGEN.com or can be requested from
QIAGEN Technical Services or your local distributor.
Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Agenda
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Principles and advantages of real-time, multiplex PCR
Critical factors for successful real-time, multiplex PCR
Application data – QuantiFast Multiplex Kits
General considerations for real-time, multiplex PCR
Checklist for successful multiplex, real-time PCR
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Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Agenda
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Principles and advantages of real-time, multiplex PCR
Critical factors for successful real-time, multiplex PCR
Application data – QuantiFast Multiplex Kits
General considerations for real-time, multiplex PCR
Checklist for successful multiplex, real-time PCR
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Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Principles and advantages of real-time, multiplex PCR
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Principles of real-time, multiplex PCR:
 Simultaneous quantification of several targets in the same reaction
 Sequence-specific probes labeled with a distinct fluorescent dye and a quencher moiety
 Can be performed as a one-step or two-step reaction
Benefits:
 Conserve precious samples – more data per sample
 Coamplification of internal controls – increased data reliability
 Increase throughput – more targets analyzed per run
 Efficient use of real-time cycler capacities – more results per run
 Save on reagent costs – targets are amplified together instead of separately
Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Agenda
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Principles and advantages of real-time, multiplex PCR
Critical factors for successful real-time, multiplex PCR
Application data – QuantiFast Multiplex Kits
General considerations for real-time, multiplex PCR
Checklist for successful multiplex, real-time PCR
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Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Critical factors for successful real-time, multiplex PCR
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Prerequisites for successful multiplexing
Successful multiplexing requires careful experimental design
and optimization. Multiplex assay optimization can be tedious
and time consuming as several factors need consideration:
 Primer concentration
 Mg2+concentration
 DNApolymerase
 dNTP concentration
 Buffer composition
Multiplex reactions must run with the same efficiency as their
singleplex reactions, even with varying targets' abundance.
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Critical factors for successful real-time, multiplex PCR
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QuantiFast Multiplex PCR and RT-PCR Kits – for instant multiplex qPCR
success
QuantiFast Multiplex RT-PCR protocolQuantiFast Multiplex PCR protocol
Accurate resultsAccurate results
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Critical factors for successful real-time, multiplex PCR
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Proprietary QIAGEN PCR Buffer technology
QIAGEN dual cation buffer for
increased specificity. Uniquely
balanced combination of two cations,
the buffer provides stringent primer-
annealing conditions over a wider
range of annealing temperatures
and Mg2+ concentrations.
Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Critical factors for successful real-time, multiplex PCR
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Synthetic Factor MP: An innovative PCR additive
Factor MP supports macromolecular crowding:
 Displaces H2O at the template
 Increases the local concentration of primers and probes on the template
 Leads to more efficient hybridization of primer/probes to the template
 Supports the binding of polymerase to the primer–template complex
 Stabilizes specifically bound primers
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Critical factors for successful real-time, multiplex PCR
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Fast cycling facilitated by Q-Bond
Q-Bond mediated fast cycling. A) Q-Bond the DNA polymerase and primer to bind as a
single complex, reducing the annealing time to a few seconds. In addition, the unique buffer
composition supports the melting of DNA, reducing denaturation and extension times.
B) Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing
annealing time.
B
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A

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Critical factors for successful real-time, multiplex PCR
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Well proven HotStarTaq Plus enzyme and blend of Omniscript and Sensiscript
HotStarTaq Plus DNA Polymerase
 Unique chemical modification of recombinant Taq DNA polymerase
 Fast 5 minute polymerase activation by initial heat incubation step
 Robust reactivation independent of PCR environment (pH, salts)
Special blend of RT enzymes for efficient and sensitive multiplex RT-PCR
 Optimized combination of Omniscript and Sensiscript
 High sensitivity
 Efficient cDNA synthesis in just 20 minutes
Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Agenda
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Principles and advantages of real-time, multiplex PCR
Critical factors for successful real-time, multiplex PCR
Application data – QuantiFast Multiplex Kits
General considerations for real-time, multiplex PCR
Checklist for successful multiplex, real-time PCR
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Application data – QuantiFast Multiplex Kits
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Wide linear range
Reliable duplex PCR
Duplex, real-time two- step RT-PCR was
carried out using the QuantiFast Multiplex
PCR Kit and self-designed TaqMan assays
for IL8 (interleukin 8) andACTB (β-actin).
Analysis of ten-fold dilutions of leukocyte
cDNA template from 100 ng to 1 pg
provided high PCR efficiencies of around
95%. The Cq values were comparable with
those achieved in control singleplex
reactions, demonstrating the reliability of
the duplex assay.
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Application data – QuantiFast Multiplex Kits
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Highest sensitivity
Sensitive duplex PCR, down to 10 copies of template. Duplicate reactions were run on the Applied
Biosystems 7500 Fast System using a DNA template mix providing 108 copies of β-actin (data shown in
insets) and 106 to 10 copies of RPS27A (a ribosomal protein). The QuantiFast Multiplex PCR +R Kit showed
higher sensitivity than the duplex PCR kit from Supplier AII, enabling the cycler in fast-cycling mode to detect
10 copies of target and quantify over 6 log dilutions of template.
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Application data – QuantiFast Multiplex Kits
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Efficient detection of targets varying greatly in abundance
Efficient, sensitive duplex analysis of targets
varying greatly in abundance. Duplex, real-
time one-step RT-PCR using in vitro transcripts of
HSP90AA1 (109, 107, 105, 103 or 10 copies) and
GAPDH (109 copies) was performed (colored
curves). For comparison, singleplex RT-PCR was
also carried out (gray curves). Duplicate
reactions were run on the QuantiFast Multiplex
RT-PCR Kit and self-designed TaqMan assays.
Reliable duplex RT-PCR is demonstrated by the
evenly spaced curves for HSP90AA1 and
overlapping curves for GAPDH. The efficiency of
HSP90AA1 amplification was in an optimal
range.
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Application data – QuantiFast Multiplex Kits
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Precise discrimination of small differences in template amount in
singleplex and duplex PCR
Linear Cq values over twofold decreases in template. Duplex and singleplex PCR were carried out using the
QuantiFast Multiplex PCR +R Kit and assays for the t(8;14) chromosomal translocation and for GAPDH.
Quadruplicate reactions were run using genomic DNA from the Ramos cell line as template (two-fold dilutions
from 10 ng to 0.625 ng). Cq values increased linearly by 1 Cq value with decrease in template dilution for both the
singleplex and duplex reactions, demonstrating the ability of the kit to precisely discriminate between small
differences in template amount.
Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Agenda
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Principles and advantages of real-time, multiplex PCR
Critical factors for successful real-time, multiplex PCR
Application data – QuantiFast Multiplex Kits
General considerations for real-time, multiplex PCR
Checklist for successful multiplex, real-time PCR5
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General considerations for real-time, multiplex PCR
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Designing primers and probes
 Keep amplicon size small: ideally 60-150 bp
 Use specialized software to design primers and probes
 Use the same settings for all assays to ensure they work optimally under
the same cycling conditions
• Amplicons should be ±5 bp and with similar GC content
• Primers and probes should have a Tm within ±5°C
 Check primer specificity using a BLAST search
 To avoid gDNA detection, design intron/exon spanning primers
Design of intron/exon spanning primers.
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General considerations for real-time, multiplex PCR
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Choice of reporter dyes
 Probes must be labeled with reporter dyes whose fluorescence spectra are well
separated or exhibit only minimal overlap
 Reporter dyes and quenchers must be compatible with the detection optics of
your cycler
 Refer to the instrument user manual for which reporter dyes can be used
Dyes commonly used in multiplex, real-time PCR
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General considerations for real-time, multiplex PCR
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Handling and storing primers and probes
 Primers and probes should be purchased from an established manufacturer
 Upon receipt, resuspend the lyophilized primers and probes and check
their concentrations by spectrophotometry
 Dissolve primers and probes in TE buffer to make a 100 µM stock solution
 Prepare small aliquots to avoid repeated freezing and thawing
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General considerations for real-time, multiplex PCR
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Evaluating the performance of a real-time, multiplex PCR assay
 Check each set of primers and probe in individual PCR assays before
combining in a multiplex assay
 Test assay performance: Assay serial dilutions of a sample containing the
target nucleic acids (n.a.)
 Test dynamic range: Make dilutions of one target n.a. keeping the
concentration of the others constant (Target n.a. cloned in a plasmid or
prepared as a PCR product can be used)
 Test for linearity : Perform reactions with ten-fold dilutions of template, and
check if the Cq values are similar to those of the corresponding single PCR
assays. A standard curve can be used to evaluate the linear range and the
PCR efficiency of the assay.
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General considerations for real-time, multiplex PCR
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Programming the real-time cycler
 Activate filters or detectors for the reporter dyes used in the multiplex PCR assay
 Follow the optimized cycling protocols in the handbooks, even for assays where
cycling conditions have already been established using a different kit or reagent.
Rotor-Gene Q real-time cycler
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General considerations for real-time, multiplex PCR
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Analyzing data from a real-time, multiplex PCR assay
Optimal analysis settings for each reporter dye are prerequisite for
accurate quantification data:
 Adjust the analysis settings for every reporter dye channel in every run
 The instrument software default analysis settings may not provide
accurate results and may need to be adjusted
 Save the multiplex reactions after amplification to check the PCR
products on a gel or on the QIAxcel
QIAxcel system
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Agenda
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Principles and advantages of real-time, multiplex PCR
Critical factors for successful real-time, multiplex PCR
Application data – QuantiFast Multiplex Kits
General considerations for real-time, multiplex PCR
Checklist for successful multiplex, real-time PCR
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Checklist for successful multiplex, real-time PCR
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Design primers and probes
 Choose predesigned assays (predesigned or published in literature)
 Carefully design assays yourself reporter dyes and quenchers
Choose reporter dyes and quenchers
 Choose appropriate combination of reporter dyes
 Use non-fluorescent quenchers
Choose reagents
 Optimize reaction conditions
 Use dedicated Master Mix (e.g., QuantiFast Multiplex PCR and RT-PCR kits)
Reconstitute primers and probes
 Use TE to prepare a 100 µM stock solution
 Store frozen in small aliquots away from light
Set up your real-time cycler
 Check if your cycler needs to be calibrated for the reporter dyes and activate the filters
 Adjust the analysis setting for each reporter dye channel in each run
Evaluate the performance of the multiplex assay
 Check that primer-probe set works in singleplex PCR
 Compare the performance in Multiplex PCR
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Versatility of QIAGEN's QuantiFast Multiplex kits
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Optimized ROX concentration, universal protocol
Rox Dye Kits Compatible cycler
In Master Mix
QuantiFast Multiplex PCR Kit (80) 204652
QuantiFast Multiplex PCR Kit (400) 204654
QuantiFast Multiplex PCR Kit (2000) 204656
QuantiFast Multiplex RT-PCR Kit (80) 204852
QuantiFast Multiplex RT-PCR Kit (400) 204854
All cyclers from Applied Biosystems
except Applied Biosystems 7500,ABI
ViiA7
Separatetube
QuantiFast Multiplex PCR +R Kit (80) 204752
QuantiFast Multiplex PCR +R Kit (400) 204754
QuantiFast Multiplex PCR +R Kit (2000) 204756
QuantiFast Multiplex PCR +R Kit (4000) 204757
QuantiFast Multiplex RT-PCR +R Kit (80) 204952
QuantiFast Multiplex RT-PCR +R Kit (80) 204954
QuantiFast Multiplex RT-PCR +R Kit (80) 204956
 Applied Biosystems 7500,ABI
ViiA7†
 Bio-Rad, Cepheid, Eppendorf,
Roche and Stratagene/Agilent‡
QIAGEN
*ROX dye must be added to master mix. †
ROX dye not required.
One for all protocol: optimized cycling conditions for 2-plex/3-plex/4-plex and universal
primer/probe concentrations!
Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Thank you for attending
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Contact QIAGEN Technical Service
Call: 1-800-426-8157 for US
Call: +49 2103-29-12400 for EU
www.support.qiagen.com
Laura Alina Mohr, MSc.
laura.mohr@qiagen.com
Questions?
Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017