Real-Time PCR Workshop

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Real-Time PCR Workshop

• Why Real-time PCR? Advantages and Disadvantages
• Theory of Real-time PCR
• Types of Real-time PCR Quantification
• Choosing Housekeeping Gene for Normalization

All information are adapted from relevant
websites as qPCR using real-time detections are
developed by different companies. Please read
carefully and follow protocols from the kits you
use.
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Disadvantage of traditional PCR

• Why Real-time PCR ?

ABI Real-Time PCR vs Traditional PCR (www)

• Poor precision (Northern could even be better)
• Low sensitivity
• Short dynamic range lt 2 logs
• Low resolution
• Non-automated
• Size-based discrimination only
• Results are not expressed as numbers
• Ethidium bromide staining is not very
  quantitative
• Competitive (mimic) PCR is laborious and
  tedious (very labor intensive)

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• Why Real-time PCR ?

Advantages of real-time PCR

• amplification can be monitored real-time
• wider dynamic range of up to 1010-fold
• 10,000 to 100,000-fold more sensitive than RNase
  protection assay, 1000-fold more sensitive than
  dot blot hybridization
• (i.e. requirement of 1000-fold less RNA than
  conventional assays)
• no post-PCR processing of products
• (No gel-based analysis at the end of the PCR
  reaction ? high throughput)
• ultra-rapid cycling (30 minutes to 2 hours)
• highly sequence-specific

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• Why Real-time PCR ?

wider dynamic range
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• Why Real-time PCR ?

Advantages of real-time PCR

• amplification can be monitored real-time
• wider dynamic range of up to 1010-fold
• 10,000 to 100,000-fold more sensitive than RNase
  protection assay, 1000-fold more sensitive than
  dot blot hybridization
• (i.e. requirement of 1000-fold less RNA than
  conventional assays)
• no post-PCR processing of products
• (No gel-based analysis at the end of the PCR
  reaction ? high throughput)
• ultra-rapid cycling (30 minutes to 2 hours)
• highly sequence-specific

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Disadvantages of real-time PCR

• Why Real-time PCR ?

• Requires expensive equipments and reagents
• Intra- and inter-assay variations
• Due to its extremely high sensitivity, you may
  get high deviations of the same treatment,
  optimal benefit from the above advantages
  requires a clear understanding of the many
  options available for running real-time PCR
  experiment
• ? theory of real-time PCR

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2. Theory of real-time PCR
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2. Theory of Real-time PCR

• Linear ground phase
• PCR is just began
• Fluorescence emission at each cycle has not yet
  risen above background
• Baseline fluorescence is calculated at this time

PCR can be broken into 4 major phases
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2. Theory of Real-time PCR

• Log-linear phase
• PCR reaches its optimal amplification period with
  the PCR doubling after each cycle in ideal
  reaction conditions

• Plateau phase
• The plateau stage is reached when reaction
  components become limited and the fluorescence
  intensity is no longer useful for data
  calculation

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2. Theory of Real-time PCR
The five-fold dilution series seems to plateau at
the same place even though the exponential phase
clearly shows a difference between the points
along the dilution series. This reinforces the
fact that if measurements were taken at the
plateau phase, the data would not truly represent
the initial amounts of starting target material.
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2. Theory of Real-time PCR
What is CT (threshold cycle)?
The Amplification Plot contains valuable
information for the quantitative measurement of
DNA or RNA. The Threshold line is the level of
detection or the point at which a reaction
reaches a fluorescent intensity above background.
The threshold line is set in the exponential
phase of the amplification for the most accurate
reading. The cycle at which the sample reaches
this level is called the Cycle Threshold, CT.
These two values are very important for data
analysis using the 5 nuclease assay.
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What is DRn?
2. Theory of Real-time PCR

• Rn is the Rn value of a reaction containing all
  components (the sample of interest)
• Rn- is the Rn value detected in NTC (baseline
  value)
• DRn is the difference between Rn and Rn-. It is
  an indicator of the magnitude of the signal
  generated by the PCR
• DRn is plotted against cycle numbers to produce
  the amplification curves and gives the CT value

Rn
Sample
?Rn
Threshold
Rn
-Rn
No Template Control
CT
cycle number
0
10
20
30
40
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3.Types of real-time PCR quantificationa
Detectionb Calculationc Normalization (in
part4 )
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• Detection chemistries
• Four common ways
• DNA binding dyes
• E.g. SYBR Green
• Hydrolysis probes
• TaqMan
• Hybridisation probes
• E.g. Light cycler
• Hairpin probes
• Molecular beacons

3. PCR Quantification
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3. PCR Quantification

• SYBR Green (double-stranded DNA binding dye)
• At the beginning of amplification, the reaction
  mixture contains the denatured DNA, the primers,
  and the dye. The unbound dye molecules weakly
  fluoresce, producing a minimal background
  fluorescence signal which is subtracted during
  computer analysis.
• After annealing of the primers, a few dye
  molecules can bind to the double strand. DNA
  binding results in a dramatic increase of the
  SYBR Green I molecules to emit light upon
  excitation.
• During elongation, more and more dye molecules
  bind to the newly synthesized DNA. If the
  reaction is monitored continuously, an increase
  in fluorescence is viewed in real-time. Upon
  denaturation of the DNA for the next heating
  cycle, the dye molecules are released and the
  fluorescence signal falls.

Mapping Protein/DNA Interactions by Cross-Linking
(NCBI Books) (www)
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3. PCR Quantification
SYBR Green emits a strong fluorescent signal
upon binding to double-stranded DNA
nonspecific binding (primer dimer) is a
disadvantage check your reactions on gel
first! requires extensive optimisation
requires melting point curve determination
longer amplicons create a stronger signal,
amplicons should be 100 to 200 bp in size
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3. PCR Quantification
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The TaqMan 5 exonuclease assay
FRET Förster/fluorescence resonance energy
transfer DNA Polymerase 5' exonuclease activity
Mocellin et al. Trends Mol Med 2003 (www)
3. PCR Quantification
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FRET
ABI Real-Time PCR vs Traditional PCR (www)
3. PCR Quantification
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The TaqMan 5 exonuclease assay In addition to
two conventional PCR primers, P1 and P2, which
are specific for the target sequence, a third
primer, P3, is designed to bind specifically to a
site on the target sequence downstream of the P1
binding site. P3 is labelled with two
fluorophores, a reporter dye (R) is attached at
the 5 end, and a quencher dye (D), which has a
different emission wavelength to the reporter
dye, is attached at its 3 end. Because its 3
end is blocked, primer P3 cannot by itself prime
any new DNA synthesis. During the PCR reaction,
Taq DNA polymerase synthesizes a new DNA strand
primed by P1 and as the enzyme approaches P3, its
5 3 exonuclease activity processively degrades
the P3 primer from its 5 end. The end result is
that the nascent DNA strand extends beyond the P3
binding site and the reporter and quencher dyes
are no longer bound to the same molecule. As the
reporter dye is no longer in close proximity to
the quencher, the resulting increase in reporter
emission intensity is easily detected.
Human Molecular Genetics 2. NCBI Books (www)
3. PCR Quantification
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Probe sequences are not altered by PCR, so they
can still be used in a subsequent assay, e.g.,
for mutation detection or SNP analysis
A The donor-dye probe is labeled with fluorescein
at the 3 end and the acceptor-dye probe is
labeled with LightCycler Red at the 5 end.
Hybridization does not take place during the
denaturation phase of PCR and, thus, the distance
between the dyes is too large to allow energy
transfer to occur.
B During the annealing phase, the probes
hybridize to the amplified DNA fragment in a
close head-to-tail arrangement. When fluorescein
is excited by the light from the LED, it emits
greenfluorescent light, transferring the energy
to LightCycler Red, which then emits red
fluorescent light. This red fluorescence is
measured at the end of each annealing step, when
the fluorescence intensity is highest.
Light cycler
C After annealing, the temperature is raised and
the HybProbe probe is displaced during
elongation. At the end of this step, the PCR
product is double-stranded and the displaced
HybProbe probes are again too far apart to allow
FRET to occur.
https//www.roche-applied-science.com/sis/rtpcr/ht
c/htc_fst/010500.jsp
3. PCR Quantification
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Comparing three different fluorescence-monitoring
systems for DNA amplification.
Wittwer, 1997 (www)
3. PCR Quantification

• Depends on the accumulated amplification product
• Depends on the 5-exonuclease activity of the
  polymerase
• Depends on the independent hybridization of
  adjacent donar and acceptor probes

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Molecular Beacons

• Molecular beacons are the simplest hairpin probe
  flanked by 2 inverted repeats.
• Reporter and quencher dyes are attached to each
  end of the molecule, causing a reduction in
  fluorescence emission in hairpin formation
• When bound to the target, the quencher and
  reporter are separated, allowing reporter
  emission.

Thermodynamic stability Probe-target helix gt
hairpin structure gt mis-matched probe target helix
3. PCR Quantification
Mocellin et al. Trends Mol Med 2003 (www)
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3.Types of real-time PCR quantificationa
Detectionb Calculationc Normalization (in
part4 )
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3. Types of real-time PCR quantification
Absolute quantification
Relative quantification (relative fold
change) i. Relative standard method
ii. Comparative CT (2 -??CT) method
3. PCR Quantification
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ASHI Quarterly
3. PCR Quantification
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3. PCR Quantification
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Absolute quantification

• This method assumes all standards and samples
  have approximately equal amplification
  efficiencies
• More labour-intensive
• because of the necessity to create reliable
  standards for quantification
• include these standards in every PCR
• 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
• Accurate determination of total RNA concentration
  is particularly important
• quantification by OD measurement faces problem of
  DNA contamination or inaccurate results from the
  spectrophotometer
• RNA constituting on average only 50-80 of the
  purified nucleic acid
• Additional step of DNase removal should be
  carried out prior to any RT step

3. PCR Quantification
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3. Types of real-time PCR quantification

• Absolute quantification
• Relative quantification (relative fold
  change)
• Relative quantification determines the changes in
  steady-state mRNA levels of a gene across
  multiple samples and expresses it relative to the
  levels of an internal control RNA.
• relative quantification does not require
  standards with known concentrations
• i. Relative standard method
• ii. Comparative CT (2 -??CT) method

3. PCR Quantification
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Relative standard curve method

• 1. Construct a relative standard curve

3. Divide the amount of c-myc by the amount of
GAPDH to determine the normalized amount of c-myc
(c-mycN).
2. Calculate the input amount by entering the
following formula in an adjacent cell 10
cell containing log input amount
3. PCR Quantification
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Types of real-time PCR quantification
Absolute quantification
Relative quantification (relative fold change
vs calibrator) i. Relative standard curve
method ii. Comparative CT (2 -??CT) method
3. PCR Quantification
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Validation experiment for comparative CT method
ABI-7700 User Bulletin 2
3. PCR Quantification
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Comparative CT method-I
ABI-7700 User Bulletin 2
3. PCR Quantification
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Comparative CT method - II
ABI-7700 User Bulletin 2
3. PCR Quantification
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3. PCR Quantification
control
D Ct target - ref
ref control
D Ct 9.70
target control
av 19.93
av 29.63
D Ct target - ref
experiment
target treated
D Ct -1.7
ref treated
Difference DCt-DCt DDCt (-1.7) -9.70
-11.40
av 18.03
av 19.80
Exercise By 2 ??CT, fold change???
2702
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4. Housekeeping Gene for Normalization
None of the identified reference for data
normalization are ideal.

• Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
• b -actin
• Ribosomal RNA (rRNA) 28S, 18S

Housekeeping Gene for Normalization
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GAPDH
4. Housekeeping Gene for Normalization
(Bustin, 2000)
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GAPDH concentrations vary
4. Housekeeping Gene for Normalization

• between different individuals (Bustin et al.
  1999),
• during pregnancy (Cale et al. 1997),
• with developmental stage (Puissant et al. 1994,
  Calvo et al. 1997),
• during the cell cycle (Mansur et al. 1993),
• apoptosis (Ishitani et al. 1997)
• food deprivation (Yamada et al. 1997)
• Inducer
• after the addition of the tumour promoter
  12-Otetradecanoyl-phorbol-13-acetate (Spanakis
  1993),dexamethasone (Oikarinen et al. 1991) and
  carbon tetrachloride (Goldsworthy et al. 1993).
• Insulin stimulates GAPDH transcription (Rolland
  et al 1995, Barroso et al. 1999)
• calcium ionophore A23187 induces GAPDH
  transcription
• Growth hormone (Freyschuss et al. 1994), vitamin
  D (Desprez et al. 1992), oxidative stress (Ito et
  al. 1996), hypoxia (Graven et al. 1994, Zhong
  Simons 1999), manganese (Hazell et al. 1999) and
  the tumour suppressorTP53 (Chen et al. 1999),
  have all been shown to activate its transcription
• retinoic acid (Barroso et al. 1999) downregulate
  GAPDH transcription in the gut and in adipocytes,
  respectively.
• unregulated in cancer
• in rat hepatomas (Chang et al. 1998),
• Malignant murine cell lines (Bhatia et al. 1994)
  and
• Human prostate carcinoma (Ripple Wilding 1995)

Its use as an internal standard is
inappropriate It is a mystery why GAPDH continues
to find favor as an internal standard.
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b-actin concentrations vary widely in response to

• experimental manipulation in human breast
  epithelial cells (Spanakis 1993)
• in various porcine tissues (Foss et al. 1998)
  canine myocardium (Carlyle et al. 1996)
• the presence of pseudogenes interferes with the
  interpretation of results (Dirnhofer et al. 1995,
  Raff et al. 1997, Mutimer et al. 1998)
• primers commonly used for detecting -actin mRNA
  amplify DNA (Dakhama et al. 1996).

4. Housekeeping Gene for Normalization
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Common normalizing housekeeping gene

• Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
• b -actin
• Ribosomal RNA (rRNA)
• 28S, 18S
• Varying ratios of rRNA to mRNA have been reported
  (Solanas et al.,2001)
• Use random hexamer instead of oligo dT in the RT
  step

4. Housekeeping Gene for Normalization
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Optimization of Primers equal Tm (58-600 C)
15-30 bases in length GC content 30-80 no
runs of four or more Gs (any nucleotide) no
more than two GC at the 3 end no G at the 5'
end amplicon size 50-150 bp (max 400) span
exon-exon junctions in cDNA to avoid genomic DNA
being amplified
ABI Primer Express Software Tutorial (www)
4. Housekeeping Gene for Normalization
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Optimization of primers (Dissociation curve)
4. Housekeeping Gene for Normalization
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FINAL REMARKS Doing real-time RT-PCR is easy,
but making real time RT-PCR data a meaningful
figure of qPCR mRNA expression profiles should
have some more careful considerations such as

• Different detection chemistry fit different
  purposes
• Make a dilution of a template, either sDNA, sRNA
  or total RNA for a standard curve
• Correlation coefficient of the standard curve gt
  0.99?
• Normalization of samples obtained experiments can
  be carried out against 2 or 3 housekeeping genes
  whose expression has been shown to be unaffected
  by experimental conditions.

Good luck with your experiments and with your
career!
4. Housekeeping Gene for Normalization
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References

• Wong, M.L. and Medrano J.F. 2005 Real-time PCR
  for mRNA quantitation. Biotechniques 39(1)
  75-85.
• Bustin, S.A. 2002 Quantification of mRNA using
  real-time reverse transcription PCR (RT-PCR)
  trends and problems. J Mol Endocrinol 2923-39.
• Bustin, S.A. 2000 Absolute quantification of mRNA
  using real-time reverse transcription polymerase
  chain reaction assays. J Mol Endocrinol
  25169-193.
• Websites from ABI, MJ companies, etc.