A practical approach to assay design for qPCR

A Practical Approach to
Assay Design for qPCR
Overcoming Difficult Assays, Designs and
Optimization while Conforming to the MIQE
Guidelines

Francisco Bizouarn
International Field Application Specialist
Gene Expression Division
Bio-Rad Laboratories

A new beginning.
AMPLIFICATION

www.bio-rad.com/genomics/pcrsupport

What is MIQE? It’s a Checklist
AMPLIFICATION

• qPCR community driven
guidelines for essential and
desired information in litterature;
– Experimental Design
– Sample Information
– Nucleic Acid Extraction
– Reverse Transcription
– qPCR Target Information
– qPCR Oligonucleotides
– qPCR Protocol
– qPCR Validation
– Data Analysis


Generating a good assay is easy
AMPLIFICATION

• Following a few simple steps:
– Design assay

– Run a gradient

– Run a dilution series to validate
assay dynamic range

• Meeting MIQE guidelines requires
very little additional effort.
– Target Information

– Oligonucleotide information
– Protocol

– qPCR Protocolalidation


Assay design
AMPLIFICATION

• Often oversimplified by the use of software or by
many companies that offer design services.

• Design a critical parameter.

• Following a few simple steps will increase the
chances of designing a successful assay.

• Let’s use an example: target CCL26 in HUVEC cells


CCL26 cDNA sequence
AMPLIFICATION

CTGGAATTGA GGCTGAGCCA AAGACCCCAG GGCCGTCTCA GTCTCATAAA
AGGGGATCAG GCAGGAGGAG TTTGGGAGAA ACCTGAGAAG GGCCTGATTT
GCAGCATCAT GATGGGCCTC TCCTTGGCCT CTGCTGTGCT CCTGGCCTCC
CTCCTGAGTC TCCACCTTGG AACTGCCACA CGTGGGAGTG ACATATCCAA
GACCTGCTGC TTCCAATACA GCCACAAGCC CCTTCCCTGG ACCTGGGTGC
GAAGCTATGA ATTCACCAGT AACAGCTGCT CCCAGCGGGC TGTGATATTC
ACTACCAAAA GAGGCAAGAA AGTCTGTACC CATCCAAGGA AAAAATGGGT
GCAAAAATAC ATTTCTTTAC TGAAAACTCC GAAACAATTG TGACTCAGCT
GAATTTTCAT CCGAGGACGC TTGGACCCCG CTCTTGGCTC TGCAGCCCTC
TGGGGAGCCT GCGGAATCTT TTCTGAAGGC TACATGGACC CGCTGGGGAG
GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG TTCATAGAGT
TGACTTGTTC AT


Sequence Alignment (BLAST)
AMPLIFICATION

• Prior to designing primers, it’s
a good idea to run a
sequence homology analysis.
(BLAST)

• This allows the identification
of sequences that may co-
amplify or interfere with our
intended target.

• The data is freely available,
so why not make use of it.

• http://blast.ncbi.nlm.nih.gov


CCL26 with homologous sequences
AMPLIFICATION

TGACTTGTTC AT


2nd structure analysis of CCL26
AMPLIFICATION

• DNA is often seen as a linear
polymer.

• In it’s single stranded state
(cDNA) regions that have
complimentary sequences will
tend to hybridize generating
hairpins that may inhibit
primer annealing.

• Avoiding these sequences
when possible will improve
amplification effiecency.

• http://mfold.bioinfo.rpi.edu/cgi-bin/dna-
form1.cgi


CCL26 with 2nd structures
AMPLIFICATION

TGACTTGTTC AT


Amplicon size
AMPLIFICATION

• Classic qPCR rules dictate that amplification products be
between 75 and 200 bp in length.

• These limits are not absolute. It is better to design a larger
amplicon than to risk target specificity and primer annealing
issues

• New “ultra fast” reagents allow much larger amplicons to be
used in qPCR.


Design primers
AMPLIFICATION

• Some primer design packages will
take both sequence homology and
secondary structure issues into
account when designing assays.

• Due to the restrictions imposed on
the design software, they can fail.

• Although not recommended,
designing assays by “thumb” can be
performed.
GCGGAATCTT TTCTGAAGGC TACATGGACC
• There are also databases of freely
available primers and probes that
have been previously tested.


qPCR Target Information
AMPLIFICATION


qPCR Oligonucleotides
AMPLIFICATION


CCL26 primer design
AMPLIFICATION

TGACTTGTTC AT


Using Thermal Gradients
AMPLIFICATION

• Thermal optimization is often the first parameter an individual
using PCR will test to get the optimal reaction conditions.

• Unfortunately many qPCR users often ignore this parameter, as
though antiquated, in favor of more elaborate primer design
software packages.

• Finding the correct annealing temperature at which to run an
assay is critical.


Using Thermal Gradients
AMPLIFICATION

• 40 wells @ 5 ul each • Prepare a master-mix for 40
wells

• 100 ul 2X Supermix • Primer concentration typically
between 200 and 500nM
• ul forward primer (300nM)
• ul reverse primer (300nM)
• ul DNA or cDNA • Critical parameter: amount of
DNA or cDNA used. Use as little
• ul H20 as possible.
---------
• 200 ul total

Vortex!

Assay optimization
AMPLIFICATION

For 1 Rev 1
5’ 3’
For 2 Rev 2

For 1 For 2
Rev 1 Rev 2

10o above
design
{
5o below
design


Gradient analysis
AMPLIFICATION

CCl26 amplified using Bio-Rad iQTM SYBR® Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis


Gradient analysis
AMPLIFICATION

CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: 5ul Assay 95oC 60sec / 50x95oC 10 sec 55-70oC 60 sec / melt analysis


Optimal Annealing Range
AMPLIFICATION



Effect of Annealing Temp on C(t)
AMPLIFICATION

C(t) vs Annealing Temp

72

70

68

66
Annealing Temp

64

62

60

58

56

54

52
25 30 35 40 45 50 55
C(q)



Different reagents behave very differently
AMPLIFICATION

C(t) vs Annealing Temp C(t) vs Annealing Temp

72 72

70 70

68 68

66 66
Annealing Temp

Annealing Temp
64 64

62 62

60 60

58 58

56 56

54 54

52 52
25 30 35 40 45 50 55 25 30 35 40 45 50 55
C(q) C(q)

CCl26 amplified using Bio-Rad Sso Fast EVA Green Supermix: CCl26 amplified using Other Reagent A: 5ul Assay
5ul Assay98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt 95oC 5min / 50x 95oC 15 sec 55-70oC 60 sec / melt analysis
analysis

C(t) vs Annealing Temp C(t) vs Annealing Temp

72 72

70 70

68 68

66 66
Annealing Temp

Annealing Temp
64 64

62 62

60 60

58 58

56 56

54 54

52 52
25 30 35 40 45 50 55 25 30 35 40 45 50 55
C(q) C(q)

CCl26 amplified using Other Reagent B: 5 ul Assay CCl26 amplified using Other Reagent C: 5ul Assay
95oC 20sec / 50x 95oC 3 sec 55-70oC 30 sec / melt analysis 95oC 20sec / 50x 95oC 3 sec 55-70oC 30 sec / melt analysis


How did they fare?
AMPLIFICATION

CCl26 amplified using Bio-Rad SsoFastTM EVAGreen® Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis


Assay Validation
AMPLIFICATION

• Assays must be validated to ensure target specificity, dynamic
range and sensitivity.

• Specificity can be initially established using melt curve analysis
but subsequently need to be confirmed using sequencing or
another confirmatory tool.

• Dynamic range should cover the real life experimental range the
assay will cover.

• If an assay needs to discriminate small differences, the assay’s
capability to do so must be demonstrated.

• Additionally, very low copy and detection assays need to be
validated using tools such as Poisson distribution analysis.


Validation of dynamic range and sensitivity
AMPLIFICATION

• Confirming dynamic range of an
assay is as simple as generating
a sequential dilution series and
generating a standard curve.

• Dynamic range of assay should
encompass the range of interest.

• There is very little use in having
standard curve with a dynamic
range spanning 8 orders when all
the samples are within 10 fold of
one another.


Large dynamic range
AMPLIFICATION

1/10 1/10 1/10 1/10 1/10 1/10 1/10 1/10
Blank

10^9 copies 10^7 copies 10^5 copies 10^3 copies 10 copies

10^8 copies 10^6 copies 10^4 copies 100 copies


Large dynamic range
AMPLIFICATION

GAPDH amplified using Bio-Rad SsoFast EVAGreen Supermix: 20ul Assay 98oC 30sec / 50x 95oC 1 sec 60oC 1 sec / melt analysis


High sensitivity assay
AMPLIFICATION

1/2 1/2 1/2 1/2 1/2 1/2 1/2 1/2
Blank

25 ng / well 6.25 ng / well 1.56 ng / well 390 fg / well 98 fg / well

12.5 ng / well 3.13 ng / well 781 pg / well 195 fg / well


High sensitivity assay
AMPLIFICATION

CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 58oC 5 sec / melt analysis


Standard Curve
AMPLIFICATION

CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 58oC 5 sec / melt analysis


qPCR Protocol
AMPLIFICATION


qPCR Validation
AMPLIFICATION


AMPLIFICATION

• Successful assay Design

• Conformance with MIQE
guidelines

• Confidently move forward
with experiments


Parameters for Consideration
AMPLIFICATION

Sometimes a little additional optimization is
required
• Primer concentration
• 2nd structures on template
• AT rich regions
• Multiple assays on plate
• Amplicon Size
• Sequence homology
• Inhibitors


Primer Titration
AMPLIFICATION

• Primer concentration plays an important role in qPCR
amplification.

• Typical concentrations go from 200nM to 500nM but can vary
from 50nM to 800nM and sometimes higher.

• High primer concentrations dramatically increase the incidence
of non specific amplification and primer-dimers.

• Reasonably well designed assays work best at normal primer
concentrations


100nM each Primer
AMPLIFICATION

CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98oC 30sec / 50x 95oC 1 sec 55-70oC 5 sec / melt analysis


100nM each Primer
AMPLIFICATION

Replicates Mean C(t) : 27.24

Standard Deviation : 0.284



200nM each Primer
AMPLIFICATION



300nM each Primer
AMPLIFICATION



400nM each Primer
AMPLIFICATION



600nM each Primer
AMPLIFICATION



800nM each Primer
AMPLIFICATION



300nM each Primer - Optimal
AMPLIFICATION





Melt curve
AMPLIFICATION



2nd Structures on template
AMPLIFICATION

TGACTTGTTC AT

Maintain forward primer at 200nM Titer reverse primer


200nM forward -- 100nM reverse
AMPLIFICATION



AMPLIFICATION





2nd Structures on template
AMPLIFICATION

• When working with a region of DNA known to have a secondary
structure; it can be advantageous to increase the concentration
of that primer, all the while maintaining the normal primer at
regular levels.

• Caution must be used when using high primer concentrations to
avoid nonspecific amplifications.

• When working with sequences rich in secondary structures,
designing primers with higher annealing temperatures, 65oC and
above, should be considered as the higher temperatures will
help dissociate some of the structures.


AT rich sequences on template
AMPLIFICATION

TGACTTGTTC AT

Maintain forward primer at 200nM Titer reverse primer


Running Multiple assays on the same plate
AMPLIFICATION

• There is often a need to run multiple different assays
on the same plate.

• Assays should run under optimal conditions; with the
proper annealing conditions.

• Adjusting primers and conditions can help solve
these issues.


AMPLIFICATION

Different sized primers targeting same amplicon

16 16

30 30


AMPLIFICATION

Primer length – 16 bases


AMPLIFICATION



Running Multiple assays on the same plate
AMPLIFICATION

• Primer size can affect annealing dynamics.

• When annealing range is too low, primer concentration can be
increased, or primer size can be increased.

• When annealing range is too high, primer size can be reduced.

• When increasing primer concentrations, as always specificity for
the target must be evaluated.


Large amplicons
AMPLIFICATION

• Classic qPCR rules dictate that amplification products be
between 75 and 200 bp in length.

• New “ultra fast” reagents allow much larger amplicons to be
used in qPCR.

• Extending the size of the amplicon should be considered when
trying to circumvent secondary structures, sequence homology
and unfavorable regions.

• Proper validation is required.


Large amplicons – dynamic range
AMPLIFICATION

•B-Actin 1076 pb amplicon from plasmid

•109 to 10 copy per well 10 fold dilution
109 copies series

•5 ul asay run on CFX384 using Bio-
Rad’s SsoFast EVA Green Supermix
10 copies
•Protocol : 98oC 3 min
45 x 95oC 1 sec 66oC 5 sec
melt curve


Large amplicons - sensitivity
AMPLIFICATION

•B-Actin 1076 pb amplicon from plasmid

•105 to 200 copy per well 2 fold dilution
series
105 copies
•5 ul asay run on CFX384 using Bio-
Rad’s SsoFast EVA Green Supermix
200 copies
•Protocol : 98oC 3 min
45 x 95oC 1 sec 66oC 5 sec
melt curve


Sequence Homology
AMPLIFICATION

• Designing primers on a region of template sequence
homologous to another gene should be avoided if
possible.


AMPLIFICATION

TGACTTGTTC AT

Poor specify is likely outcome


AMPLIFICATION

TGACTTGTTC AT

Increased specify


AMPLIFICATION

TGACTTGTTC AT

With very difficult targets


Sequence Homology
AMPLIFICATION

• Designing primers on a region of template sequence
homologous to another gene should be avoided if possible.

• When inevitable, a single primer can be designed to anneal on a
homologous region for a series of genes. The other primer
should be annealing on a clean region or one that has no
homology with genes annealed by the first primer.

• Multiple primers should be designed and tested.

• If a single primer anneals multiple to targets, it will generate a
linear amplification of DNA, where as, if both primers anneal, the
amplification will be exponential.


Inhibitors
AMPLIFICATION

• PCR although a routine process, is an elegant dance, comprised
of a series of complex processes and interactions between
enzymes, primers, nucleotides, template DNA and buffer
components.

• Inhibition can be caused by various chemicals, solvents, ions and
peptides (to name a few).

• Since their presence is never uniformly distributed in samples,
they cannot easily be corrected for in the reaction mix. They
should be removed from the sample (as possible), or a supermix
that can withstand this inhibitory effect should be used.


Blood Serum
AMPLIFICATION

<2.5 %

10 %

CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay
98oC 30sec / 50x95oC 1 sec 60oC 5 sec / melt analysis


Blood Serum
AMPLIFICATION

<0.0098 %
0.039 % <0.0089%

0.039%

CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: CCl26 amplified using Other Reagent A: 5ul Assay
5ul Assay 95oC 3 min / 50x 95oC 10 sec 60oC 60 sec / melt 95oC 5min / 50x 95oC 15 sec 60oC 60 sec / melt analysis

<0.0089%
<0.0089%
0.039%
0.039%

CCl26 amplified using Other Reagent B: 5ul Assay CCl26 amplified using Other Reagent C: 5ul Assay
95oC 20sec / 50x 95oC 3 sec 60oC 30 sec / melt analysis 95oC 20sec / 50x 95oC 3 sec 60oC 30 sec / melt analysis


Understanding your assay
AMPLIFICATION


Speed - SsoFast
AMPLIFICATION

SsoFast EvaGreen Supermix

Sso7d from Sulfolobus solfataricus
– 7kD, 63 aa.
– Thermostable (Tm >90°C)
– No sequence preference
– Binds to dsDNA (3-6 bp/protein molecule)
– Monomeric

• Minimal inhibition of PCR by use of
EvaGreen

• Higher activity

• Tolerant to PCR inhibitors


Throughput
AMPLIFICATION

• The CFX384 real-time PCR
detection system brings flexibility
and ease of use to researchers
performing high-throughput real-
time PCR in a 384-well format.

• With up to 4-target detection,
unsurpassed thermal cycler
performance, and powerful, yet
easy-to-use software, the CFX384
system has been designed for the
way you work.
– FAST – shorten the time from
experiment setup to results
– FRIENDLY – a new standard for
ease of use, delivering data you
can trust with no maintenance
– FLEXIBLE – customize a set up
that fits individual laboratory needs


Conclusions
AMPLIFICATION

• The key to successful qPCR experiments lie with proper design,
optimization and validation.

• qPCR assay optimization and dynamic range validation require
very little time and effort and help guarantee that the results will
be reproducible and comparable form experiment to experiment.

• Implementation of MIQE guidelines is almost seamless.

• If potentially interfering elements are discovered at the design
and optimization phases, they can be accounted for and
possibly corrected.

• Designing good assays does not have to be a “chore”, it can be
quite fun!


AMPLIFICATION

• Thank You!

• Questions?


A practical approach to assay design for qPCR

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