Fast qPCR assay optimization and validation techniques for HTS

Fast qPCR assay
optimization and validation
techniques for HTS

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

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

• A little extra effort in the beginning
will make a tremendous amount of
difference in the analysis when the
assay is run hundreds or
thousands of times.

www.bio-rad.com/genomics/pcrsupport

Assay design
AMPLIFICATION

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

• 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.


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.


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(t)



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(t) C(t)

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(t) C(t)

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



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



Melt curve
AMPLIFICATION



Increased primer concentrations can increase
nonspecific amplification products generated.
AMPLIFICATION

200nM Socs1 primers Conditions: 95oC 3min / 50x 95oC 10 sec 55-70oC 30 sec / melt analysis

400nM Socs1 primers Conditions: 95oC 3min / 50x 95oC 10 sec 55-70oC 30 sec / melt analysis


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

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


2nd Structures on template
AMPLIFICATION

TGACTTGTTC AT

Maintain forward primer at 200nM Titer reverse primer


200nM forward -- 100nM reverse
AMPLIFICATION



AMPLIFICATION





AT rich sequences on template
AMPLIFICATION

TGACTTGTTC AT

Maintain forward primer at 200nM Titer reverse primer


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 bp 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 bp 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.

• When inevitable, a single primer can be designed to anneal on a
homologous region for a series of genes. The other primer
should 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 targets, it will generate a
linear amplification of DNA where as if both primers anneal, the
amplification will be exponential.


Inhibitors
AMPLIFICATION

Blood Serum

<2.5 %
10 %

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


Inhibitors
AMPLIFICATION

Blood Serum

<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


Scale effect on reproducibility
AMPLIFICATION

45
6
40
5

35
4

delta C(t)
40 pg
X
C(t)

30 1 pg
X
3
delta

25
2

20 1

15 0
2.5 5 10 20
Volume (ul)

CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 2.5-20 ul Assay 98oC 30sec / 50x 95oC 1 sec 60oC 5 sec / melt analysis


Quality reagents are scalable
AMPLIFICATION

45 45
6 6

40 40
5 5

35 35
4 4

delta C(t)
40 pg 40 pg

delta C(t)
C(t)

C(t)
30 1 pg 30 1 pg
3 3
delta delta

25 25
2 2

20 1 20 1

15 0
15 0
2.5 5 10 20
2.5 5 10 20
Volume (ul)
Volume (ul)

CCl26 amplified using Bio-Rad iQ SYBR Green Supermix: CCl26 amplified using Other Reagent A: 2.5-20 ul Assay
2.5-20 ul Assay 95oC 3 min / 50x 95oC 10 sec 60oC 60 sec / melt 95oC 5min / 50x 95oC 15 sec 60oC 60 sec / melt analysis

45 45
6 6

40 40
5 5

35 35
4 4
delta C(t)

delta C(t)
40 pg 40 pg
C(t)

C(t)
30 1 pg 30 1 pg
3 3
delta delta

25 25
2 2

20 1 20 1

15 0 15 0
2.5 5 10 20 2.5 5 10 20
Volume (ul) Volume (ul)

CCl26 amplified using Other Reagent B: 2.5-20 ul Assay CCl26 amplified using Other Reagent C: 2.5-20 ul Assay
95oC 20sec / 50x 95oC 3 sec 60oC 30 sec / melt analysis 95oC 20sec / 50x 95oC 3 sec 60oC 30 sec / melt analysis


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


Speed
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


Conclusions
AMPLIFICATION

• The key to speeding up any screening process begins with
proper design and optimization.

• 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.

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

• As demands for shorter run times increase, proper care in the
selection of reagents and instruments is required.


AMPLIFICATION

• Thank You!


Fast qPCR assay optimization and validation techniques for HTS

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