Quantitative PCR (qPCR) has become a common tool in the

lab, and advancements in speed and throughput have

made this technology more attractive to those looking for a
quick answer. However, the quality of data generated by
qPCR is heavily dependent upon efforts spent in
qPCR Assay Optimization for
Multiplexing: Practical Advice for
Improving Data Quality
qPCR is heavily dependent upon efforts spent in
developing and optimizing the assays for their best
performance. This session will outline typical assay design
challenges, with solutions, and provide advice towards
developing multiplex qPCR assays that have all the
performance of an assay run in single-plex, while utilizing
your instruments full potential for sample through-put and
data generation.
qPCR Assay Optimization for Multiplexing: Practical
Advice for Improving Data Quality
Scott D. Leppanen
Sr. Field Applications Scientist
Boston, Northeast NA
Scott.Leppanen@Agilent.com
QPCRjust put in the nucleic acids and go, right?
Performance can be good right out of the box
Less emphasis on assay validation/optimization
Does the assay fit your experimental needs?
Will the results lead to accurate data interpretation? Will the results lead to accurate data interpretation?
How much work is required to get the assay to a point
where I can run my valuable samples and get reliable
data?
will I have the informational leverage to make the right decisions?
Typical cycle of qPCR Assay Development
Assay
Standard curve
With negative
controls
No
Primer optimization
Matrix; Redesign?
Primer
Specificity?
Good
%Efficiency?
Quantitative range
adequate?
Multiplex Development?
Normalizer Stability?
yes
yes
Re-validate format
Design Primers
Analyze precious
samples
Re-validate format
For optimal Multiplex PerformanceValidate assays
in Singleplex
qPCR Assay Design and Optimization Ensures High Data
Quality and Easy Transition to Multiplex
Validation of primer and detection chemistry performance
easiest in singleplex
Efficiently designed, robust performing assays in singleplex will Efficiently designed, robust performing assays in singleplex will
very likely translate to same in multiplex
Its highly recommended to evaluate in singleplex and build from
there..
MIQE guidelines, encourages improved assay design
and optimization
Suggests reporting of Limit of Detection (LOD) and assay
Efficiencies
LOD=concentration that can be detected with reasonable (>95%) certainty
Show that assay Efficiency and LOD are not impaired by
the inclusion of assays run in multiplex
Intends to make qPCR data interpretation more reliable
Why Optimize?First few qPCR Cycles
Limited Target with Vast Excess of Primers
Primer Dimer Specific Amplicon
Taq
0HOW
>>[Primers]
gDNA,
Alternative
Sequences,
Accurate
Quantification
Compromised
Detection
0HOW
$QQHDO
([WHQG
'HWHFW
Sequences,
Matrix Effects
Detection Chemistry..Start
Simple with SYBR
Detection chemistry is not the largest determinant in assay quality parameters
such as sensitivity, LOD, or specificity
Ultimate decision can be economic/convenience
Have transient need to screen lots of genes> DNA binding dye
Have persistent desire to assay fewer genes, lots of samples>multiplex probes
Quality qPCR data is driven by the reliability and specificity
of priming; if you dont make it you will not detect it!
qPCR Assay Primer & Chemistry Strategy
Choose a small amplicon (100-150bp) that contains sequence
that is compatible with probe based chemistry
Plan to optimize using SYBR chemistry
Constrain the Tms of primers to approx. 60C
Optimize on the basis of efficiency and specificity
Order just primers, work out conditions, run samples until
Sybr chemistry limits utility of PCR or multiplex is desired
Order probe and confirm measuring range of assay.
Optimizing for Assay Sensitivity and Specificity
Run standard curves and look for linearity and good Efficiency
To determine that the reactions are specific, turn to the melt
curve of a DNA binding dye assay
Generally, specific primers that are not distracted with creating
non-specific products yield an Efficiency ~100%
Assay will be more sensitive and give better detection resolution
A qPCR assay that has a robust amplification and melt curve
and no evidence of Primer Dimer will perform better in
Multiplex
DNA + monomer
weak fluorescence
dsDNA Binding Dyes in qPCR
PCR amplification
Multimerizes on dsDNA
dye is fluorogenic
Temperature
DNA Binding Dye Detection, Built in Specificity
Amplification
of sampleswhat products
contribute to the signal ?
No
Template
Standard
Curve
Template
Negative vs Positive qPCR Controls
One Peak?
Template
Control
(NTC)
No
Template
Standard
Curve
Template
Negative vs Positive qPCR Controls
One Peak?
Template containing sample should
have narrow peak at high (~80C), with
high signal
NTC should be flat, no Ct
Template
Control
(NTC)
NTC
Negative vs Positive qPCR Controls
One Peak or Two?
Primer Dimer will show as a shorter, wider
peak at ~70C, will form inversely to
[template]
Template
NTC
Template
added
Beyond Melting Curve Analysis:
Agilent BioAnalyzer
TM
Instances when SYBR melt data is
misleading
Why? SYBR Green in assay concentration
is non-saturating & shows evidence of
sequence specific binding
BioAnalyzer can provided leverage for
troubleshooting troubleshooting
BioAnalyzer evaluates oligo length
Electropherogram provides accurate high
resolution information about what is being
made in qPCR tube
Hubert Zipper, et al
Nucleic Acids Res. 2004; 32(12): e103.
Troubleshooting a positive NTC with similar Tm, is it contamination?
Beyond Melting Curve Analysis:
Agilent BioAnalyzer
TM
Size: 21 + 51 bp
NTC
BioAnalyzer eGram suggests dimer rather than contamination
Your First qPCR Assay..You Need Control
Materials
Positive control material should reflect intended sample
complexity and matrix, Good Examples.
Any prep that has your gene of interest, not necessary to be experimental
Spike plasmid or PCR product into cDNA background
Numerous examples exist where samples did not perform the same as a
plasmid or PCR product alone
Stratagene Universal Reference RNA, human, Stratagene Universal Reference RNA, human,
mouse or rat
Negative controls provide reference of assay bounds
Zero template is not often zero signal/noCt in qPCR
Include NTC (Primers alone) as well as NoRt (RNA prior to RT)
Your First qPCR Assay.. qPCR Standard Curve
A Tool for qPCR Validation
1:5 Serial dilution of cDNA,
7-10 pts in triplicate
~300nM each primer
SYBR Green MM
Standard Curve
Efficiency
E =10
[-1/slope]
-1
Quantity
C
t
LogQuantity
C
t
E =10
[-1/slope]
-1
Expect:
Good linear fit (R
2
> 0.98)
High efficiency (E = 90 110%)
Log transform
Stratagene Brilliant III SYBR MM
Eff=105.6%
R
2
=0.97
Where does the assay fail, where is the sweet spot, or quantification cut-off at the high
and low end of expression?
Can I detect replicates with precision? What is the resolution of the assay?
Does the final assay range have a good %E?
qPCR Standard Curve
Tool for qPCR Validation
R =0.97
Eff=105.0%
R
2
=1.0
qPCR Standard Curve
Tool for qPCR Validation
Determines acceptable range of assay Cts for downstream sample analysis, ie Rel Quant
My Standard Doesnt fit my Need
Low sensitivity, Low Efficiency, Short range
Change annealing times and temperatures to favor the lower Tm primer
Longer time and lower temp
Add more of both primers
Re-assay at a greater resolution, 2 fold dilutions
Poor Precision, High Efficiency (>100%) Poor Precision, High Efficiency (>100%)
Investigate different primer design, move primers 1-3 bases
Re-assay primers at different use concentrations
Not always both primers that are promiscuous
Limit the bad primer and salvage the pair
Primer Optimization Matrix
Determine an indiviualized Ideal [Primer]
1. Mid-range [RNA] plate
2. Vary forward and reverse
concentrations in combination
3. Compare rxn condition Cts
and melt curve products
Forward [Primer]
75 150 300 nM
75
150
25.1 26.4 25.7
26.3 25.3 23.3
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4. Select pairs with no loss of
Ct, no dimer
Goal:
Select Forward and Reverse ratio that yields lowest Ct
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reaction with no non-specific products
Future Multiplex qPCR should be easier to set up
300
nM
24.6 23.4 23.5
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Is Multiplex qPCR Right for You?
Singleplex..
transient
assay
needs, few
samples
Multiplex..
persistent
assay
needs, lots
of samples
Multiplex qPCR is the amplification of two
or more targets in the same reaction
Microarray validation
(many genes w/few samples, 1x validation) (many genes w/few samples, 1x validation)
Gene expression sample screening
(a few genes w/many samples)
Small gene expression validation
(few genes w/few samples)
Diagnostics applications
(Few targets w/consistent throughput)
Benefits of Multiplexing
Reduces the number of reactions required for each
sample to generate data sets..
Reduces sample requirement per gene
Faster to data, more efficient use of
instrumentation instrumentation
In optimized assays, lower variation across assays
because assays share conditions in multiplex
Saves on reagent costs, plastics, etc
Requires instrument capable of multiplexing.
Capabilities and limitations, is dye calibration required, is there cross-talk
across channels
Design may be difficult and optimization is often required
Drawbacks of Multiplexing
Probe chemistry is more expensive
Competition between individual assays for the reaction components due
to expression differences in assays
High probability of unwanted cross-oligo interactions
Probability of Oligo Interaction is Cause for
Concern
Forward
Reverse
Probe
Single Assay
In a single probe-based assay, there are 3 possible oligo interactions
Duplex Assay
Forward
Reverse
Probe
Probability of Oligo Interaction is Cause for
Concern
In a duplex assay, there are 15 possible oligo interactions, 105 in a 5-plex assay
Forward
Reverse
Probe
In Silico Multiplex
Primer/Probe Design
Test assays in singleplex
Multiplex Design and Optimization Flow

Run and Analysis
Experiment Design
Optimize Primer concentrations
Test Assays in Multiplex
Primer limitation
Reagent optimization
TaqMan

Probe or Other Probe Technology

required to Multiplex, example TaqMan
Unbound probe free in
solution
Probe and primer bind target, Fluor
Energy
Dissipation
Donor dye
(Reporter)
Acceptor dye
(Quencher)
Light
Energy transfer
Taq
Probe and primer bind target, Fluor
quenched
Taq
Taq extends and hydrolyzes probe,
fluor free to emit fluorescence -->
indicate accumulation of amplicons
Taq
Light Emission Light
TaqMan Probe Design for Multiplex
Considerations and Guidelines
length short as possible, <30bp
Tm = primer Tm + 10C 2C
LNA or MGB bases best for SNP detection & AT rich amplicons
Taq
LNA or MGB bases best for SNP detection & AT rich amplicons
Use dark quencher
Avoid G on 5 end, adjacent fluor, Quenching A<T<C<G
Minimize number of Gs in probe sequence, <50% G + C
No more than 10bp away from extending primer (no less than 2bp)
Primer Design for Multiplex
Taq
Considerations and Guidelines
length 20-30bp, shorter is better
Tm = 60C
Less than 2C frwd and rev
OH
Less than 2C frwd and rev
G/C clamp on ends
Avoid secondary structure, consult M-Fold for amplicon
Multiplex Design and Optimization Software
Beacon Designer will Design multiplex assays
Checks all primer/primer and primer/probe interactions for up to 4 targets in
a multiplex reaction
http://www.premierbiosoft.com/
BioSearch Technologies Real Time Designer
Free online, upload sequence or accession numbers, select Free online, upload sequence or accession numbers, select
instrument, easy to use, multiplex capable
http://www.biosearchtech.com/ProbeITy/design/inputsequences.aspx
IDT Oligo Analyzer has sophisticated tools to evaluate primers
and probe cross hybridization
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
M-Fold for oligo secondary structure, BLAST for specificity
Probes Fluorescent Wavelength Range-
Ensure no Cross Talk
Quartz-tungsten halogen lamp excitation range (350-750nm)
TAM TET TxRed JOE
FAM HEX ROX Cy5 Alx350 Cy3
350nm 700nm
- Many other fluorescent dyes can be detected using a given filter sets
- Be aware of potential for cross-talk between selected dyes
Ex.
Dye Selection is Instrument Specific.. The
Agilent Mx uses Filters
Em.
PMT
Emission
Gain Setting
for each
Filter range ~10nm band Filter range ~10nm band- -pass pass
To optimize for sensitivity and specificity, the excitation and
emission filters of the Mx are selectively optimized to, minimize
background and cross-talk while enhancing dye discrimination.
Signal (RFU)
for each
channel
Ensure that your Instrument has no Signal Cross
Talk
1500
2000
2500
3000
RFU
FAM
HEX
350 nm 750 nm
Excitation Filter
Agilent Mx.. <1% signal Leak from Adjacent Wavelengths
-500
0
500
1000
1500
RFU
FAM HEX ROX Cy5
HEX
ROX
Cy5
Excitation Filter
10 nm Interference
Em. Filter
In Silico
Primer/Probe Design
Test assays in singleplex
Multiplex Design and Optimization

Already optimal from
initial assay design
Run and Analysis Experiment Design
Optimize Primer concentrations
Test Assays in Multiplex
Primer limitation
[Probe] optimization

C
t
Log quantity
GOI
C
t
Norm
Validation in Single-plex Format.. is there any
Loss in Performance? Iterate improvement
Log quantity
GOI +
Norm
C
t
Log quantity
Try each assay pair-wise, to determine compatibility with others
swap out reagents, change concentrations, reevaluate performance
C
t
mulitplex
singleplex
Validation of Multiplex Assay.. There should
be no Loss in Performance
C
t
Log quantity
Acceptable limits:
Efficiencies within 5-10%, similar LOD and sensitivity
Ct less than ~1Cycle difference between formats
Four-plexed qPCR Standard Curve, Find Problem
Assay and limit Probe, Example
~180% efficiency
- CY5 primer/probe
interacting with other
reactions to generate
additional signal
Determine Minimal Probe Concentration
Required for Robust Ct, Limit for Problem assay
Cy5 Assay:
In this example, 150nM probe is
adequate, 100nM
is not robust, 50nM is limiting,
in detection of high [Std]
Primer Limitation when Targets of Different
Expression Compete for Reagents
Dominant Target in the multiplex reaction uses up reagents
Other assays will perform better when dominant limited
Lower primer concentrations of dominant, change dR,
not Ct; A>B
Free up reagents to other assays without changing Ct or Free up reagents to other assays without changing Ct or
Efficiency
Cycle #
F
l
u
o
r
e
s
c
e
n
c
e
dR
A
B
C
Increase qPCR Components when Targets of
Different Expression Compete for Reagents
Performing multiplex assay it is necessary to
increase reaction component concentration
Increase Taq concentration (>50-100% up to 3.5U).
Increase dNTP concentration (50-100%).
Increase Mg
++
concentration (1-5 mM).
In some cases, increase buffer concentration to 1.5x
increase total volume of reaction mix to 50uL
Brilliant Multiplex qPCR Master Mix
Increased concentration of reagents
(polymerase, Mg
2+
, dNTPs, etc) optimized
for multiplex reactions
SureStart hot-start DNA polymerase
Allows for multiplexing of up to 5 targets with Allows for multiplexing of up to 5 targets with
Probes or Beacons
Greatly reduces optimization and increases
success rate of multiplex assays
Success! Five Target Standard Curves
500ng-1.95ng qPCR Reference RNA + Alien cDNA spiked in using 4-fold dilutions. .
ALEXA 350 DYE = ENOS, E= 96.6%
ROX DYE = CFTR,E= 110.1%
HEX DYE = HFE,E= 110.3%
FAM DYE = ALIEN, E= 78.2%
CY5 DYE = CYCLOPHILIN, E= 104.0%
Capitalizing on Specific PCR and Detection on the
Agilent Mx qPCR
Detection of 8 pathogens with Molecular Beacons on Agilent MX3000p,
4 color instrument
Dual priming strategy; 8 unique Beacon probe detection, individual fluors
or combinations of fluors, 8 unique fluor characteristics, detect in 4 plex
Validated vs and more sensitive than culture, singleplex=multiplex
Multicolor Combinatorial Probe Coding (MCPC)
Approach used different fluors
for same oligo probe
Output of detection is a
composite of fluor
detection
Universal amp primer> no
bias in amplification
Equivalent
detection
across
formats
Organism Gene Dye(s) Quencher
S. aureus Nuc FAM Dabcyl
L. monocytogenes Hly FAM&HEX Dabcyl
Salmonella Typhi ssaR ROX Dabcyl
Shigella ipaH Cy5 Dabcyl
E. coli O157:H7 rfbE HEX Dabcyl
V. cholerae ctxA ROX&HEX Dabcyl
V. parahaemolyticus tdh FAM&Cy5 Dabcyl
S. pyogenes spy1258 FAM&ROX Dabcyl
same probe sequence
Conclusion- Assay Optimization for Multiplex qPCR
Determine if multiplex assay is advantageous for your
experimental needs..a SYBR assay may be adequate
Optimize in singleplex format to determine the minimum, optimal
performing concentration, to reduce the probability of dimer
formation
Rely upon the Standard curve and the SYBR melt curve to guide
development
Try full multiplex combination, iterate performance pairwise Try full multiplex combination, iterate performance pairwise
Limit primers of dominant PCR if necessary in multiplex
Increasing reagent concentration will allow easier optimization of
multiplex qPCR reactions>consider commercial reagents
Confirm that assay performance does not suffer in multiplex
Agilent is a qPCR system provider, with instruments and reagents
that perform accurately and sensitively in multiplex format.
Agilent qPCR technical and informational
Thank you for your
Attention, I look
forward to your
questions
Agilent qPCR technical and informational
support:
Email: QPCR@Agilent.com
1 (800) 227-9770, x3, x4, x3
Agilents Stratagene qPCR System
5 Channel qPCR
instrument Mx3005p
Scanning optics
Full featured software Full featured software
Compatible with all
chemistry and reagents
Filters can be mismatched
to support alternate dyes
(ie DSF method)
Mx Optical System
PMT
Halogen white
light bulb
Excitation filter
wheel
Emission filter
wheel
Coaxial fiberoptic
cable
96 well plate
1 blank filter
position for
excitation calibration
Read head scans over each well,
filter wheels rotate between scans
Passive Reference Fluor ROX is Optional on MX
Channel may be needed for Multiplex
Passive Reference Fluor (ROX) spiked into qPCR master
mix at outset of assay setup
Rox corrects for sample volumes, plasticware inconsistency,
bubbles
Instrument control of uniformity
Improves data by reducing variation (%CV) among technical
replicates replicates
Optional, not required, SW allows for data with and without Rox
normalization
Rox channel can be used for multiplex, perform a true 5 plex