EGT Genomics 2004

1
Real-time qPCR Theory, advantages How to get
started Cynthia Potter Field Applications
Specialist Eurogentec, North America
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Real-time qPCR

• Real-time PCR Theory, advantages (PCR defined)
• Data and how to read it
• Reverse Transcription and real-time qPCR
• Analysis methods
• Applications
• Other Fluorescence systems
• Getting Started

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Real-time qPCR

• Real-time PCR Theory, advantages (PCR defined)
• SYBR Green
• 5nuclease assay (TaqMan)
• FRET defined
• how it works
• multiplex
• when and why
• dark quenching
• Data and how to read it
• amplification curves
• Ct, Rn, delta Rn, baseline, background, plateau,
  exponential
• melt curves
• what it tells us, what it doesnt tell us
• standard curves
• specifically for your instrument (MX 3000p)
• efficiency
• how to determine it
• how to achieve it

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Real-time qPCR

• Reverse Transcription and real-time qPCR
• theory
• methods
• one-step
• two-step
• Analysis methods
• relative quantitation
• absolute quantitation
• Applications
• detection
• gene expression
• allele discrimination
• SNP-single nucleotide polymorphism
• Other Fluorescence systems
• MGB LNA
• Molecular Beacons
• FRET probes
• Scorpion

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Real-time qPCR

• Getting Started
• picking a fluorescence system for your experiment
• defining GOI
• picking a control
• good design
• extraction storage-products, what to expect
• lab protocol for prevention of contamination
• RNase and DNase is not just a state of mind
• what kit should I use? Ingredients and what they
  are there for
• Hot start Taq why
• dNTPs (w/ and w/o dUTP)
• UNG
• RT-why MmLV?
• Passive reference
• MgCl2
• ratios (Taq, dNTPs, Mg)
• stabilizers and how they affect the reaction
• optimization-when and why

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Real-time PCR Theory, advantages (PCR defined)
Normal, typical, regular, gel-based
PCR vs. real-time qPCR
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Typical PCR

• denaturation 94ºC
• annealing 55ºC-65ºC
• elongation 72ºC
• repeat 30-40 times

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Phases of amplification

• Exponential phase
• Plateau phase

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Gel analysis of PCR product

• Size
• Yield visual or densitometry

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Disadvantages of regular PCR

• Time consuming
• PCR, gelcasting, loading, running, staining,
  computer analysis
• Endpoint detection
• Final yield is not directly related to initial
  amount of DNA, i.e. A small amount of sample DNA
  with lots of Taq and dNTPS can give the same
  plateau (endpoint) as a large amount of sample
  DNA with limiting reagents.
• Risk of contamination
• No information about purity of product

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To avoid these disadvantages a new technique was
developed Real-time qPCR
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Real-time qPCR
A PCR application in which you have the ability
to monitor or visualize the increase in PCR
product by a proportional increase in
fluorescence. From the rate of increase in
exponential phase, where reagent concentrations
are not limiting, starting quantity can be
extrapolated.
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Definitions
Ct
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Real-time PCR basics

• DNA is amplified in PCR with a fluorescence
  detection system consisting of synthetic DNA, or
  oligonucleotides (oligos)
• Real-time qPCR instrument which consists of
• an optics system for exciting fluorescent dyes
  and capturing their emission signals
• a PC to interpret these signals graphically
• a thermal cycler

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SYBR green I

• Non specific
• Binds to all dsDNA
• ?SYBR green ?fluorescence
• Used for quantitation and meltcurves

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SYBR Green

• Intercalates to dsDNA, fluoresces
• Inhibits DNA amplification so concentration is
  critical
• Detects specific and non-specific dsDNA including
  primer-dimers, and primer-primer interactions
• Can run a melt curve to look at product
  specificity

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5 Nuclease Assay

• Specific
• Binds only to gene of interest
• Double Dye Oligo hybridizes ? Taq cleaves of F ?
• ? fluorescence
• Used for quantitation and allelic discrimination

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SYBR vs. Probes
New users often pick SYBR because it seems
easier than probes cheaper than
probes Consider the following downsides to
SYBR optimization for each primer
set background is always higher for SYBR reducing
sensitivity Then consider you must have a
shorter amplicon for real-time (use software to design your oligos for
real-time So you have to re-design anyway, so
designing a probe/primer set for real-time is
just as easy as designing a primer set for
real-time SYBR because you use the same software!
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SYBR vs. Probes
Also, the savings using SYBR up-front are you
dont have to buy a probe. How much is a probe,
anyway? 10 nmoles of a probe at 200, 25uL
reaction volumes, 200nM probe i.e. very
typical yields, price, volume, concentration
10 cents/rxn at list
price for 200_at_25uL reactions SYBR qPCR MasterMix
is 23 more than Universal qPCR MasterMix, which
comes out to 11.5 cents per reaction. So, they
are the same price! SYBR can be useful for
developing a new amplicon, by melting the
amplicon instead of running a gel. However, for a
new amplicon a gel should be run for SYBR and for
probes.
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SYBR vs. Probes
Why use SYBR? Validation of microarray is the
most common use of SYBR Green MasterMix, when it
would be too costly to design a probe for every
hit that may end up being invalid.
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5 Nuclease Assay

• The first step of each PCR cycle is the
  dissociation of the two strands of the DNA from
  each other, also termed melting.
• The temperature drops from 95 to about 70, at
  which the probe anneals.
• It drops further to about 60, at which the
  primers anneal.
• The Taq Polymerase attaches to the hydroxyl
  terminus of the 3-end of the primer.
• It immediately starts extending the strand and
  encounters the probe in its path.
• The probe is cleaved off piece by piece.

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5 Nuclease Assay

• It becomes clear looking at this diagram, the
  importance of
• a probe with a melting temperature (Tm ) greater
  than that of the primer pair by 8-10C
• Tms of the primers very similar to each other
• The goal is to allow the probe to anneal to all
  the targets, saturate the sample
• When the temperature has lowered to the anneal
  temperature, which is just below the Tm of the
  primers, the primers will extend and cleave off
  the probe.
• You dont want the probe coming off simply
  because of temperature. You want the probe to
  come off because it was annealed to target and
  forced, enzymatically, off.

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FRET
A probe consists of a Fluorophore (F) and a
Quencher (Q). When the F and Q are complexed
together on the same oligo, an interaction,
termed FRET for Fluorescence Resonance Energy
Transfer or FÖrster RET, is occurring between
them. The F is a donor and the Q is an
acceptor in this process. The F is excited by
light of a certain wavelength and donates the
energy to an acceptor, the Q. The Q releases
fluorescence at its specific emission wavelength.
If the optics of the instrument are set to
capture the emission wavelength of the F, nothing
will be detected if FRET is occurring between the
F and Q. When the probe is cleaved off by the
extending primer, the F and Q will not have FRET
occurring between them because they have been
separated from each other. Then the fluorescence
emission of the F will be released and the
optics, set to detect the wavelength of the F,
will report it. Hence the use of the word
reporter for the fluorophore on a Double-Dye
Oligo.
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FRET Efficiency
If this transfer of FRET is 100 efficient, then
when the F and Q are together no fluorescence of
F is escaping, i.e. no background
fluorescence If this transfer is 80 efficient,
then there is background for the 100 efficient
transfer case, when the F and Q are separated the
system goes from 100 Q fluorescence to 100 F
fluorescence for the 80, the total fluorescence
when F and Q are separated is full F signal minus
F background. So, it is important to have highly
efficient FRET
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Efficiency of FRET depends on

• r distance between fluorophore and quencher
• J(l) overlap integral spectral overlap of
  emission of fluorophore and absorption of
  quencher.
• QD Quantum yield of fluorophore (Donor).
• (relative orientation of transition dipoles)

the r is good for probes that are 30nts or
less dont worry about 3 and 4. the overlap
integral is important. This is simply the area of
the spectra of the F and Q that intersect. This
is important because this value varies between
the dyes available on the market.
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TAMRA as a quencher

• Why is TAMRA the most common dye in use today?
  FAM and TAMRA were the sequencing dyes in use at
  the time real-time was invented. In short, it was
  what was lying around.
• TAMRA has limited spectral overlap with FAM, TET,
  HEX, JOE, VIC (Yakima Yellow)
• TAMRA is fluorescent, so it contributes to
  background.
• Now we know that the efficiency will be low when
  TAMRA is the quencher, and, in addition, TAMRA
  has native fluorescence which will also
  contribute to background...

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Dark Quenching
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Dark Quenching
The answer to this problem is to use dyes as
quenchers that have maximal spectral overlap with
each reporter. Since there are several reporters
possible, and for many assays, it is required to
multiplex, the best option would be to have many
quencher dyes, picked solely for their overlap
with each reporter dye. This would not work if
each quencher was fluorescent like TAMRA because
it would be a mess of spectra. The answer is to
use a dark quencher, where dark means the dye
has no native fluorescence.
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Spectra of Dark Quenchers
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Other Advantages of Dark Quenching
Calibration of an instrument for the spectrum of
a quencher is unnecessary, since subtraction of
this spectrum from the overall fluorescence is
not needed multiple dark quenchers can be used
in a single multiplex multiplexing is easier,
because user can pick reporters that are
spectrally resolved from each other
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Molecular Beacon

• Specific
• Binds only to gene of interest
• MB hybridizes ?
• F and Q separated ?
• ? fluorescence
• Mainly used for allelic discrimination

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Scorpions

• Specific
• Binds only to gene of interest
• Primer binds ?
• DNA elongation ?
• compl. sequence ?
• probe hybridizes ?
• ? fluorescence
• Mainly used for allelic discrimination

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Hybridization Probes

• Specific
• Bind only to gene of interest
• Both probes bind ?
• F1 and F2 in each others vicinity ?
• FRET occurs ?
• ? fluorescence
• Mainly used for allelic discrimination in
  combination with meltcurve

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Reverse Transcription
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What is one-step two-step qRT-PCR?
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What is one-step two-step qRT-PCR?
Two step qRT-PCR Reverse transcription of RNA
sample as first step used oligo dT and/or random
nonamers/hexamers. cDNA can be stored for future
use or used as target for specific primers in
real-time qPCR. It provides more flexibility in
that the cDNA can be used for more than one qPCR
reaction and can be archived, so that it
eliminates the need to continually isolate
RNA. One step qRT-PCR RT reaction and the qPCR
are performed in the same sealed tube, without
manual transfer between reactions. The buffer is
a combination of an RT and a PCR buffer, in which
both enzymes work. The one step RT qPCR reaction
is a closed tube assay, so contamination can be
avoided. It saves pipetting steps and time and
handling is easy.
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• Which is better one-step or two-step?
• It really comes down to your needs
• throughput
• contamination
• quantity of target RNA.

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One step qRT-PCR
A one step qRT-PCR is recommended when doing HTS
(High Throughput Screening), where the experiment
should be as easy and straightforward as
possible. One step eliminates all possibility of
contamination and reduced efficiency due to
varying accuracy in transfer between the RT step
and the qPCR step. This aspect can be very
important when there are many technicians
involved with varying degrees of experience and
accuracy. It is only possible to add specific
primers and since the RT reaction is performed at
40-50C, the primers can misprime the RNA and
therefore transcribe unwanted sequences, which
then also will be amplified in the PCR step,
leading to aspecific PCR products. This
disadvantage is inherent to the method. Using a
probe may eliminate the problem, but if the
efficiency of the PCR step is so reduced, then a
probe might not detect anything.
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Two step qRT-PCR

• For the RT step, it is possible to use three
  different primer types
• Oligo d(T) primers, which bind to the poly A tail
  of the RNA and then only transcribe RNA. This
  will avoid contamination with genomic DNA. As the
  poly A tail is located at the beginning of the
  gene it will also lead to more full transcripts.
• Random nonamers, which bind anywhere in the
  genome and allow the reverse transcriptase to
  fill up the gaps, will ensure high yields.
• Specific primers, which bind to the gene of
  interest, will give specific products.
• The combination of oligo d(T) primers and random
  nonamers will give the highest yields and the
  longest transcripts, whereas specific primers
  transcribe only specific RNA but reduce the
  yield.
• For low abundance RNA, two step is a logical
  choice over one step.

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Hot start Taq why
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Hot start Taq why
When ordinary unmodified Taq sits at room
temperature (rt) with primers, template, buffer,
Mg and dNTPs it has sufficient activity to extend
primers, which are annealed incorrectly (as they
will be at 35ºC below their Tm). Any wrongly
annealed primers will be extended by the enzyme
at 1 or 2bp per minute at rt, as opposed to
2000-4000bp at 72ºC. Obviously as one goes from
rt to 95ºC there will be a race between primers
dropping off at above their Tm and the amount of
bp being added. Those primers may be annealed as
primer dimer or on the template in the wrong
place. If they extend, they will be a perfect
match at the wrong place, and amplification will
occur during all subsequent cycles. If two
primers are within say 500bp of one another then
they may give rise to up to a 500bp product etc.
This amplification will compete with
amplification of the correct product. These
aspecific products influence the results of a
real-time PCR in a negative way leading to
inaccurate quantitation. To avoid the
amplification of aspecific products, a hotstart
Taq polymerase, HotGoldStar, is used, which only
becomes active after heating at 95?C for 10
minutes.
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Use Hot start Taq!
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What is UNG?
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UNG
Uracil N-Glycosylase or Uracil DNA
Glycosylase here is the original abstract Gene.
1990 Sep 193(1)125-8. Use of uracil DNA
glycosylase to control carry-over contamination
in polymerase chain reactions. Longo MC,
Berninger MS, Hartley JL. Life Technologies,
Inc., Gaithersburg, MD 20877.   Polymerase chain
reactions (PCRs) synthesize abundant
amplification products.Contamination of new PCRs
with trace amounts of these products, called
carry-over contamination, yields false positive
results. Carry-over contamination from some
previous PCR can be a significant problem, due
both to the abundance of PCR products, and to the
ideal structure of the contaminant material for
re-amplification. We report that carry-over
contamination can be controlled by the following
two steps (i) incorporating dUTP in all PCR
products (by substituting dUTP for dTTP, or by
incorporating uracil during synthesis of the
oligodeoxyribonucleotide primers and (ii)
treating all subsequent fully preassembled
starting reactions with uracil DNA glycosylase
(UDG), followed by thermal inactivation of UDG.
UDG cleaves the uracil base from the
phosphodiester backbone of uracil-containing DNA,
but has no effect on natural (i.e.,
thymine-containing) DNA. The resulting
apyrimidinic sites block replication by DNA
polymerases, and are very labile to acid/base
hydrolysis. Because UDG does not react with dUTP,
and is also inactivated by heat denaturation
prior to the actual PCR, carry-over contamination
of PCRs can be controlled effectively if the
contaminants contain uracils in place of thymines.
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UNG

• DNA(w/T)?PCR?DNA amplicons(w/U)
• If all amplicons have uracil, then if they become
  contaminants in another PCR reaction they wont
  be amplified because
• DNA(w/T)DNA(w/U)contaminant?UNG treatment(37C)
• UNG cleaves the uracils without breaking
  sugar-phosphodiester backbone of DNA
• UNG deactivated by 95C hold for 10 min. that
  activates hotstart Taq
• DNA(w/T)DNA(w/vacant holes where U used to
  be)?PCR?DNA amplicons(w/U)
• PCR was halted for the strands that had vacant
  holes, so contaminant wasnt amplified.

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UNG-problems
Note at the end of PCR you have DNA
amplicons(w/U) sitting in UNG UNG can be still
active if temperature drops to 37C (the
temperature for the UNG reaction prior to
PCR) So, UNG can cleave up the uracils. This
cleavage makes the strands very labile to
acid/base hydrolysis and over time you can see a
degraded melt curve
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When why is UNG necessary
UNG prevents carryover contamination, so if
plates are being opened and gels run, it is
necessary. In some labs, plates are never
opened, gels never run. This is typical at a
pharmaceutical company where, if a gel was run,
it would occur in another room, by a different
technician, under conditions of extreme
cleanliness. In these cases, the use of UNG as a
necessity is arguable.
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RT-why MmLV?
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RT-why MmLV?
In real-time PCR it is recommended to use small
amplicons, without secondary structures.
Therefore it is unnecessary to use reverse
transcriptases that can transcribe long
fragments, which can lead to more secondary
structures, which, overall, are costly to the
reaction by depleting the available reactants and
lowering the efficiency. MmLV Reverse
Transcriptase gives high yields and longer
transcripts, which leads to more sensitive PCRs,
since more full copies of cDNA are
available. MMLV-RT and engineered derivatives
(Kotewicz et al. 1988) have significantly less
RNAse H activity than AMV-RT (Gerard et al.
1997). As even reduced RNAse H activity can
interfere with the synthesis of long amplicons
(DeStefano et al. 1991), MMLV-RT may be a better
choice if the aim of the experiment is the
amplification of full-length cDNA molecules. (S.
A. Bustin, 2000)
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MgCl2
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MgCl2
MgCl2 is one of the most important component
differences between regular and real-time PCR. It
is essential that the concentration is at least
3mM For Molecular Beacons standard is 3.5mM For
Double-dye oligos minimum is 5mM, Mg optimization
can be run and usually falls between 5 and
7mM. Lowering Mg to avoid misprimes is not
effective in real-time. If there are misprimes,
then oligos should be re-designed.
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Why use ROX as Passive Reference?
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Passive Reference

• A dye or dye-conjugate in Mastermix to correct
  for
• pipetting errors
• bubbles
• fingerprints
• In short, to normalize for non-PCR-related
  fluctuations in
• fluorescence signal.
• Requirements for a good Passive Reference
• must be able to excite the fluorescence and read
  the emission on real-time instrument
• stable in solution, so that distribution of PR is
  homogeneous
• cannot bind to walls or precipitate
• Fluorescence intensity must be constant
  throughout all relevant cycles of PCR (no
  ROX-drop)
• Fluorescence intensity cannot be greater than
  that of the probe, or other reporting moiety

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Passive Reference
ROX-drop is a drop in fluorescence intensity
over subsequent cycles of PCR. Its presence ruins
the entire point of having a passive reference in
the first place. Eurogentec has proprietary
ROX-conjugate that does not exhibit
ROX-drop ABI has its own ROX-conjugate that
exhibits ROX-drop
ROX-drop from Development and evaluation of
passive reference candidate compounds for
normalization of signal in real-time PCR C.
Potter, M.K. Johansson, D. Dick, R. Cook
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Passive Reference
ROX is a good dye to use, because a ROX
double-dye oligo is rather expensive, but ROX as
a passive reference is either cheap or already
included in off-the-shelf kits.
from Mx3000P software
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Stabilizers and how they affect the reaction
Stabilizers are non-reactants that should be
added to mastermix to a minimum since they can
crystallize and precipitate thereby decreasing
the well-to-well uniformity. Stabilizers vary
between manufacturers, and can shift product
Tms. Glycerol is added to help binding to
targets despite secondary structure. Glycerol
precipitates. DMSO is added to stabilize SYBR
Green dye
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Cycling
allow enough time for the reactions to occur.
This varies from instrument to instrument,
because temperature transition times vary. Make
sure you are reading at the anneal/extend step
(usually 58-60C). This should be the temperature
just below the Tm of the primers. If you are
using SYBR Green, you may be reading/acquiring at
the extend step. This is usually 72C, but it
can be any temperature above the Tm of the
primer-dimer and below the Tm of the amplicon.
Note If primer-dimer is present it is lowering
the efficiency of the reaction. Good design and
optimized concentrations will help to avoid
primer-dimer, so that reading at 72C would not
be necessary
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Optimization
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Concentration Optimization
As we have seen concentrations of oligos and Mg
concentrations can have important effects on both
probe and SYBR Green reactions Eurogentec kits
include an extra tube of MgCl2 for Mg
optimizations, if the end-user so desires A
concentration optimization can be run for SG and
probe assays. Optimal performance is achieved by
selecting the primer or probe/primer
concentrations that provide the lowest Ct-value
and the highest increase of fluorescence compared
to the background.
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Primer Optimization
Primer Optimization Matrix
Because the individual efficiency of the forward
and reverse primer can vary, their respective
concentrations must vary to compensate.
Therefore all permutations of a selected number
of primer concentrations must be tested. For
instance, there are nine possibilities of how
forward and reverse primer concentrations could
be combined for the chart above.
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Probe-primer Optimization
Figure shows the raw data of the above described
dilution series. Three different probe
concentrations and nine different primer
concentrations were combined.
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Probe-primer Optimization
Figure shows the quantitation data. The curve
with the lowest Ct-value (arrow) was chosen for
further analysis (brown curve on the left
side)   The curve with the lowest Ct value and
the best amplification should be chosen for
further experiments.
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Target Design

• The size of the amplicon is defined by the
  distance between the 5 ends of the two primers.
  When selecting the amplicon, it is better if it
  is 100bp, because they amplify more efficiently
  than longer ones
• The 3 end of the primer that is on the same
  strand as the probe should be, optimally, 5 bases
  from the 5 end of the probe, but within 10 bases
  is acceptable if the amplicon is short, this
  requirement is much easier to meet.
• Runs of four or more nucleotides are not
  recommended, especially G. The interaction
  between the nucleotides can cause the
  oligonucleotide to fold in on itself producing
  secondary structure
• It is important that there is not a 5G on the
  probe. It has been shown that guanosine quenches
  the adjacent fluorophore.
• It is recommended to pick the probe that has more
  Cs than Gs, due to the strong interaction that G
  displays with itself.

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Target Design

• It is important that the primers specifically
  anneal. The last 5 nucleotides on the 3 end of
  the primers could anneal non-specifically if
  their collective binding power is strong enough.
  This is why more than 2 Gs, 2 Cs or 1 GC is not
  recommended
• It is important to design a probe with a melting
  temperature (Tm) greater than that of the primer
  pair by 7-10C, and having the Tms of the primers
  very similar to each other (within 2C). The
  goal is to allow the probe to anneal to all the
  targets, saturate the sample, as the temperature
  ramps down to the anneal temperature of the
  primers. When the temperature is at the anneal
  temperature, below the Tm of the primers, the
  primers will extend and cleave off the probe.
  You dont want the probe coming off simply
  because of temperature. You want the probe to
  come off because it was annealed to target and
  forced off, enzymatically, separating fluorophore
  and quencher.

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Target Design

• Check the Tm s of the oligos with a separate
  program. The sequences are not longer than 30
  nucleotides so to calculate the Tm, use the
  Nearest-Neighbor method. The GC calculators
  are ineffective for oligonucleotides under 30
  nucleotides such as a probe because GC
  interactions are important in folding but the
  interactions between all bases that neighbor
  each other are not taken into account. Make sure
  the Mg is entered correctly for the assay.
  This concentration has a huge effect on the Tm.
• After designing oligos, do a BLAST search or a
  similar analysis to determine the specificity.
  Both right and left need to match to get a
  positive result
• http//www.ncbi.nlm.nih.gov/blast/Blast.cgi

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Target Design

• Choose amplicon with minimal secondary structure.
  This is important, since secondary structures
  could affect the efficiency of the reaction. In
  any real-time application it is desirable to
  obtain a 100 efficiency of the amplification
  reaction. If the secondary structure is
  thermodynamically more stable than the oligo
  target hybridization, hybridization of the target
  will be disfavored and efficiency will be
  lowered. Secondary structure could also prevent
  polymerase read through. Keep the GC content
  between 20 and 80. G-C-rich sequences are
  susceptible to non-specific interactions that may
  reduce reaction efficiency and could produce
  non-specific signal in SG assays. A recommended
  program to test for secondary structures is
  Mfold. This is a versatile folding program for
  use in analyzing amplicons, linear probes over 30
  nucleotides, and Molecular Beacons. It is found
  at the following link
• http//www.bioinfo.math.rpi.edu/mfold/dna/form1.c
  gi.

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Target Design
How to use Mfold 1. name the sequence 2. enter
sequence into large box 3. In folding
temperature enter the annealing temperature of
the reaction (60C). 4. In Ionic conditions
change the units to mM and enter the Na as
50 mM and Mg for 3-6 mM. 5. enter email (the
program sends you nothing, but this must be
entered) 6. Click the fold DNA button (next to
happy face) 7. You will get a list of structures.
A well-designed sequence will yield only one
form. The PNG form is a nice form in which to
view the structure and the melting temperature of
the structure will appear in a separate window.
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Target Design
To judge what structures are strongly favored,
look at the delta G value 10 strongly not
favored, -10 strongly favored, with approximately
linear variation of those extremes in between. If
an amplicon secondary structure is unavoidable
the primer annealing temperature should be
increased.   Data have to be acquired at
annealing temperature for probe assays
70
Designing an Assay

• Define Hypothesis
• Define Target
• Define Control
• Define Analysis Method

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The Process
Grind up the organ, filter out non-cellular
components, and solubilize in Trizol.
Purify total RNA, DNase treat, analyze by agarose
gel, and quantitate by Ribogreen.
Pick one untreated control sample to use in a
serial dilution standard curve.
Based on the Ribogreen assay s, dilute samples
to be within the standard curve.
Run qPCR assays of target and house keeping genes.
Compare the target gene to house keeping gene to
measure changes.
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Definitions

• Rn (normalized reporter) The fluorescence
  emission intensity of the reporter dye divided by
  the fluorescence emission intensity of the
  passive reference dye
• Rn R/ROX
• Rn The Rn value of a reaction containing all
  components, including the template
• Rn- The Rn value of an un-reacted sample. The Rn-
  value can be obtained from
• The early cycles of a real-time PCR run (those
  cycles prior to a detectable increase in
  fluorescence), OR
• A reaction that does not contain any template
• NTC (no template control) - A sample that does
  not contain template. It is used to verify
  amplification quality.
• NAC (no amplification control) - A sample that
  contains all reaction components except Taq.
• Baseline The initial cycles of PCR, in which
  there is little change in fluorescence signal

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Passive Reference Effects
Rn (delta Rn) The magnitude of the signal
generated by the given set of PCR conditions. The
Rn value is determined by the following formula
?Rn (Rn) (Rn-) so, ?Rn
R/ROX-baseline/ROX Threshold The average
standard deviation of Rn for the early PCR
cycles, multiplied by an adjustable factor. The
threshold should be set in the region associated
with an exponential growth of PCR
product. Threshold baseline
(x?STDbaseline) Ct (threshold cycle) The
fractional cycle number at which the fluorescence
passes the fixed threshold
Considering the fact that the Passive Reference
has an effect on Rn, it will have an effect on
baseline, ?Rn, and, ultimately, the Ct.
74
Reaction Efficiency
PCR is exponential. A PCR efficiency of 100
means for every cycle there is a doubling. The
most important aspects of the amplification curve
are the Ct and the slope of the exponential
portion. There must be a linear portion of the
line or the reaction is not exponential and
therefore inefficient. The plateau is unimportant
since a gel can give you what the plateau
provides, but no quantification can be determined
with a gel or the plateau. End-users are looking
for linearity and reproducibility. To avoid more
work down the line, it is always best to take the
time to properly design the experiment
75
Stratagene Mx3000p

• 96-well
• peltier element
• halogen optics
• single PMT (photomultiplier tube)
• SYBR Green, FAM, Joe, HEX, Yakima Yellow, ROX,
  Texas Red, CY5
• multiplex
• approx. 2 hour run time

76
Analysis Methods
From the Mx3000P real-time PCR system Manual
Quantitative PCR This experiment type uses a
standard curve to quantitate the amount of target
present in an Unknown sample with high accuracy
using a fluorescence-labeled probe for detection.
A series of Standard samples, containing
dilutions of a known amount of target, are
amplified to generate a curve that relates the
initial quantity of the specific target to the
Ct. The standard curve is then used to derive the
initial template quantity in Unknown wells based
on their Ct values. This method is sometimes
referred to as absolute quantitation or as
standard-curve quantitation in the literature.
This experiment type is also useful for
primer/probe optimization experiments in the
absence of a standard curve. Comparative
Quantitation This experiment type is a form of
relative quantitation, comparing the levels of a
target gene in test samples (referred to as
Unknowns) relative to a sample of reference
(referred to as the Calibrator). For example, the
Calibrator sample might contain RNA from
untreated cells, while the Unknowns might contain
RNA from cells treated with different agents of
interest. This experiment type provides an
efficient method for comparing levels of RNA or
DNA across samples when information about the
absolute amounts of target in any sample is not
required.
77
Analysis Methods
Absolute Quantitation 1) Create standard curve
using an in vitro transcribed standard or
synthetic standard that is quantified
absolutely by PicoGreen. 2) Fit unknowns to it
to quantify Relative Quantitation-Analysis of
gene expression of one sample relative to a
different reference sample Standard curve
method Comparative Ct method Both involve
picking a control and gene(s) of interest. These
PCRs can be run in separate wells using SYBR or
probes or in multiplex using probes.
78
Defining HKG GOI
The source of extraction and the method of
extraction of DNA or RNA must be reliable and
reproducible. Can you use a HKG (Housekeeping
Gene) for your assay? For example, what if your
GOI (gene of interest) cannot be quantified by a
relationship to a control? If you can use a HKG,
then you must evaluate its relationship to your
GOI. A good HKG is not up or down regulated by
the effect that needs studying, e.g. condition,
drug, therapy, etc. It should be expressed in the
same range as the GOI and there should be no
similar genes or pseudogenes of the control gene.
Establish linearity for the GOI and HKG, by
dilution series. Linearity is not enough, it also
must be reproducible. Establish a range in which
you can interpolate. Extrapolation must be
qualified.
79
Definitions Active Reference the signal is
generated as the result of PCR amplification. The
active reference has its own set of primers and
probe. Endogenous and exogenous controls are
examples of active references.
80
Picking a Control
Pick a Control A good control is not up or down
regulated by the effect that needs studying, e.g.
condition, drug, therapy, medium, etc. It should
be expressed in the same range as the gene of
interest and there should preferably be no
similar genes or pseudogenes of the control gene.
Endogenous control RNA or DNA that is present
in each experimental sample as isolated. By using
an endogenous control, one can normalize
quantitation of a messenger RNA (mRNA) target for
differences in the amount of total RNA added to
each reaction. Exogenous control a characterized
RNA or DNA spiked into each sample at a known
concentration. An exogenous active reference is
usually an in vitro construct that can be used as
an internal positive control (IPC) to distinguish
true target negatives from PCR inhibition. An
exogenous reference can also be used to normalize
for differences in efficiency of sample
extraction or cDNA synthesis.
81
Absolute Quantitation
Interpolation of quantity of unknowns by a
standard curves Starting with a standard
(plasmid DNA or transcribed RNA) that is
concentrated and then read at A260 values
converted to copy number using molecular weight
of DNA or RNA prep of dilution series to the
range that mimics the concentration in a
biological sample. the amplification plots
should be evenly spaced, their linear portions
parallel to each other. Note It is not a given
that this will occur. In the dilution process,
one may find that a PCR inhibitor is being
diluted, so the efficiency may be better for a
dilute sample than a concentrated one, for
example. standard curve is generated from the Ct
for each amplification curve.
82
Absolute Quantitation
The threshold (either manually or mathematically
calculated by taking the 2nd derivative) should
be just above baseline, at the beginning of the
linear portion of each amplification plot, giving
a Ct for each that should be evenly spaced If
the Cts are evenly spaced the slope (the graph of
Ct over log copy number) of the standard curve
should be 3.3. Exponential amplification 10
(-1/slope) Efficiency 10 (-1/slope) 1 Note
It is generally not possible to use DNA as a
standard for absolute quantitation of RNA,
because there is no control for the efficiency of
the reverse transcription step.
83
Relative Quantitation DDCt Method
Normalized amount of target A unitless number
that can be used to compare the relative amount
of target in different samples. Calibrator A
sample used as the basis for comparative results.
84
Relative Quantitation DDCt Method
Comparative Ct method The amount of target,
normalized to an endogenous reference and
relative to a calibrator, is given
by 2-DDCt the efficiencies of the
amplifications of the Gene of Interest (GOI) and
Endogenous Control must be matched (approximately
equal) A sensitive method for assessing if two
amplicons have the same ef?ciency is to look at
how ?Ct varies with template dilution. For a plot
of ?Ct over log input amount, the slope should be
zero (a horizontal line). If they are not, then
standard curves must be generated to quantitate.
85
Relative Quantitation DDCt Method
?Ct GOI average Ct HSKP average Ct The
calculation of ??Ct involves subtraction by the
?Ct calibrator value. This is subtraction of an
arbitrary constant, so the standard deviation of
??Ct is the same as the standard deviation of the
?Ct value. then just evaluate the expression
2-DDCt
86
Analysis Methods Relative
A new mathematical model for relative
quantitation in real-time RT-PCR Michael W.
Pfaffl (Nucleic Acids Research, 2001, Vol.
29) and others emerge all the time...
87
Relative Quantitation Standard Curve Method
The standards, whose relative dilutions are
known, and experimental samples are diluted
carefully and standard curves generated for
each. For each experimental sample, the amount
of target and endogenous reference is determined
from the appropriate standard curve. Then, the
target amount is divided by the endogenous
reference amount to obtain a normalized target
value. one of the experimental samples is the
calibrator. An example of this would be an
untreated control in a study where gene
expression level is being related to a treatment.
Each of the normalized target values is divided
by the calibrator normalized target value to
generate the relative expression levels.
88
The Process
Grind up the organ, filter out non-cellular
components, and solubilize in Trizol.
Purify total RNA, DNase treat, analyze by agarose
gel, and quantitate by Ribogreen.
Pick one untreated control sample to use in a
serial dilution standard curve.
Based on the Ribogreen assay s, dilute samples
to be within the standard curve.
Run qPCR assays of target and house keeping genes.
Compare the target gene to house keeping gene to
measure changes.
89
Relative Quantitation Standard Curve Method
Note It is possible to use a DNA standard curve
for relative quantitation of RNA. Doing this
requires the assumption that the reverse
transcription efficiency of the target is the
same in all samples, but the exact value of this
efficiency need not be known
90
Designing an Experiment
Control kits are available, but the controls
offered may not be applicable to your GOI.
Standards for a reliable assay are as follows for
your control and your GOI each reaction must be
100 efficient or really close slope 3.2 to
3.5 or 105 to 93, respectively. make sure you
have the correct sequences (check at NCBI, the
assignment of NM_ _ _ _ _ _ means it has been
sequenced). For novel sequences be sure to search
properly in NCBI databases
91
Designing an Experiment Linearity
Establish a 5 orders of magnitude range in which
unknown quantities can be interpolated You must
prove that you can extrapolate by extending
standard range into very small quantities. Once
that linearity has been established the standard
curve that is run with each plate need only
overlap with the unknowns in terms of
concentration. A typical dilution range for a
standard curve 5-log range 20,000,000
2,000,000 200,000 20,000
2,000 copies
92
Designing an Experiment
Multiplexing When multiplexing two probes,
often one control and one GOI, you must meet the
standards of a single amplification in terms of
efficiency. Run each probe in separate wells and
mixed on the same plate in the same run. The
resultant amplification curves should be
identical. If they are not, then the reactions
are affecting each other. You can increase dNTPs,
Taq and even Mg a little so that the reagents are
not as limiting and there is less
competition. If this does not work, a re-design
of the oligos is in order.
93
Measurement Parameters change
Gene of Interest
Housekeeping Gene
Equiv. ng of Steady-State mRNA
x
x
x
x
x
PPI-2458 (mg/kg)
94
Applications
95
Applications SNP
Probes with different reporters for each single
nucleotide polymorphism. Design probes as
usual, but the Tm difference with the primers
should only be about 2-3C
96
Applications Allele Discrimination
Probes with different reporters for each allele.
Us the scatterplot functions in the software as
follows The Dual color scatter plot screen
displays a scatter plot of Fluorescence or Ct for
any two dyes assigned to the same wells. Each
point represents the coordinates of the
Fluorescence or Ct for the two dyes in a single
well. The plot provides a simple method for
grouping the sample wells according to the
amplification events indicated by either single
dye (i.e. homozygous for one of two alleles) or
by both dyes (i.e. heterozygous for the two
alleles). The Final call results screen provides
a simple depiction of whether product was
accumulated for each dye in each well. A plus
sign () signifies accumulation of product, while
a minus sign () signifies that product
accumulation was not detected. Calling is based
on Ct, where is returned if the Ct is less than
the last cycle for which data was collected.
97
Technical Support
EUROGENTEC North America, Inc. Cynthia Potter 240
West 104th St. 2B New York, NY 10025 Cell
1-215-869-9583 c.potter_at_eurogentec.com www.eurogen
tec.com