Real-Time-PCR

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Real Time PCR

• M.Prasad Naidu
• MSc Medical Biochemistry, Ph.D,.

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Traditional PCR (semiquantitative)
1 2 4 8 2
denaturation 95C
annealing 60C
n cycles
extension 72C
n
Gel electroforesis
Specificity determined by 2 primers
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Traditional PCR (semiquantitative)
Plateau phase
Variable PCR Plateau (taq efficiency decreases,
reagents get limiting, decreased denaturation
efficiency, )
Amplification Plot of 96 Sample Replicates
Plateau phase
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Principles of quantitative PCR

• Monitors the progress of the PCR as it occurs (in
  real time) by reading fluorescence intensities
  after each cycle. Intensities are proportional to
  the number of amplicons generated
• Samples are characterized by the point in time
  during cycling, when amplification is first
  detected (more starting material ? sooner an
  increase in fluorescence)
• For allelic discrimination, endpoint assays are
  used

• Temperature protocol
• also gt 30 cycles
• 30 95C
• 30 60C
• 30 72C/60C

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Exponential growth phase linear part in
logarithmic graphic
1 2 4 8 2
n cycles
n
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CHEMISTRY

• SYBR green
• Taqman probes
• Molecular Beacons
• Scorpion primers

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1) SYBR green I
CHEMISTRY

• intercalating dye, binds double strand DNA
• More sensitive than EtBr
• Specificity determined by 2 primers
• No probe required (lower costs)
• Also detection of aspecific products ? melting
  curve after PCR reaction
• Only singleplex
• No allelic discrimination possible

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Dissociation Curve
CHEMISTRY
1) SYBR green I

• Dissociation Protocol can be added to the thermal
  cycling parameters
• Allows detection of non-specific products

95C 15s
95C
60C 1min
60C 20s
cycle 40
20min
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Dissociation Curve
Raw Data View
Natural decrease in SYBR fluorescence
Tm temperature when 50 dissociated
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Derivative Data View
Dissociation Curve
Tm temperature when 50 dissociated
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Dissociation Curve
Example Presence of Primer Dimers
Product
Primer dimers or aspecific product
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CHEMISTRY
2) Taqman probe
Mechanism fluorescence resonance energy
transfer (FRET)
Annealing and polymerization
Strand displacement

• Cleavage (5 nuclease activity of taq DNA
• polymerase)
• Increase of reporter signal proportional
• to amount of amplicon produced
• Removes probe from target strand

Cleavage
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CHEMISTRY
2) Taqman probe

• Two primers a fluorogenic probe determine
  specificity
• No detection of aspecific products
• No melting curve needed (faster)
• Can be used for allelic discrimination
• Multiplex
• Synthesis of different probes required for
  different sequences

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CHEMISTRY
2) Taqman probe
Multiplex reactions possible

• Reporters FAM,TET,VIC,JOE
• Quenchers TAMRA, MGB
• Passive reference ROX

normalizes for non-PCR-related fluorescence
fluctuations occurring well-to-well
(concentration or volume differences)
Emission Profiles of Various Fluorophores
? Spectral compensation necessary
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DEFINITIONS

• Baseline
• Threshold
• Rn
• Ct

Baseline Basal level of fluorescence defined
during the initial cycles of PCR (background
fluorescence). Threshold Fixed fluorescence
level set above the baseline (statistical cutoff
based upon background fluorescence). Rn
normalized Reporter signal, level of fluorescence
detected during PCR. Calculated by dividing probe
reporter dye signal by passive reference signal
(ROX). Ct threshold Cycle, PCR cycle at which
an increase in reporter fluorescence above a
baseline signal is first detected (cycle when
fluorescence crosses the threshold).
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DEFINITIONS

• Setting baseline and threshold (exponential
  growth) ? determining Ct (threshold cycle) of
  each sample
• Ct is the cycle number at which the fluorescence
  passes the threshold

EXAMPLE GRAPHIC
threshold baseline
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Advantages of using Real-Time PCR

• COLLECTS DATA IN THE EXPONENTIAL GROWTH PHASE
• REAL TIME permanent record of amplification
• INCREASED DYNAMIC RANGE of detection
• LESS RNA NEEDED Requirement of 1000-fold less RNA
  than conventional assays
• FAST No-post PCR processing
• SENSIBLE Detection is capable down to a 2-fold
  change

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APPLICATIONS

• Real time detection
• Quantitation of gene expression
• Quantitation of RNA, DNA, cDNA
• Viral quantitation
• Endpoint detection
• Allelic discrimination (SNP genotyping)
• Plus/minus studies
• Pathogen detection

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APPLICATIONS

• Real time detection
• Quantitation of gene expression
• Quantitation of RNA, DNA, cDNA
• Viral quantitation
• Endpoint detection
• Allelic discrimination (SNP genotyping)
• Plus/minus studies
• Pathogen detection

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Quantification

• Absolute quantification (result in copy number)
  virus copy number,
• 1. Calculation by standard curve
• Relative quantification (result is given as
  relative to the reference sample) gene
  expression,
• 2. Calculation by standard curve
• 3. Use of comparative Ct method

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APPLICATIONS

• Real time detection
• Quantitation of gene expression
• Quantitation of RNA, DNA, cDNA
• Viral quantitation
• Endpoint detection
• Allelic discrimination (SNP genotyping)
• Plus/minus studies
• Pathogen detection

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RNA reverse transcription
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Absolute Quantitation Standard Curve
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Standard curve
Quantify sample by spectrofotometry, make
dilution curve
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Standard curve
Ct
Ct 29.7
Log Qty
Log Qty 3.28
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Dynamic range
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Relative Quantitation Standard Curve
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Relative quantitation example

• Cells
• Basal conditions
• Treatment IL6 3h
• Treatment OSM 3h
• Define expression of gene of interest (SOCS3)
  upon treatment, relative to expression at basal
  conditions

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We need an endogenous control to normalize for
the amount of starting material in the tube !

• ß-actin
• GAPDH
• 18S and others

The perfect standard does not exist choose the
best control for your system
ratio target gene (experimental/control) fold
change in target gene (exp/control)  fold
change in reference gene (exp/control)
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Relative quantitation example

• Make dilution series of a sample
• Read SOCS3 levels of standard curve and unknown
  samples
• Read 18S levels of standard curve and unknown
  samples
• Choose a sample (cells in basal conditions) as
  calibrator
• If level SOCS3 (sample IL6 45 min)/ level SOCS3
  (sample basal conditions) 10x
• Level 18S (sample IL6 45 min)/ level 18S (sample
  basal conditions) 2x
• ? Then the level of SOCS3 after IL6 treatment is
  5x higher than at basal conditions

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Relative Quantitation ??Ct method
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??Ct method
Principle Samples that differ by a factor of 2
in the original concentration would be
theoretically expected to be 1 cycle apart.
Samples that differ by a factor of 10 (as in our
dilution series) would be 3.3 cycles apart.
Example 1 Ct(A) 30 Ct(B) 31 RQ 21 2
Example 2 Ct(A) 30 Ct(B) 33,3 RQ 23.3 10
CT (sample) - CT (basal)
Relative Quantity 2
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BUT
sample
calibrator
8 targ 4 ref
12 targ 2 ref
ratio fold change in target gene (sample) fold
change in reference gene (sample) fold
change in target gene (calibrator) fold change
in reference gene (calibrator)

• ?CT(sample) CT (Target) - CT (Reference)
• ?CT(calibrator) CT (Target) - CT (Reference)
• ? ? CT ? CT (Sample) - ? CT (Calibrator)
• Relative Quantity 2

-??Ct
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??Ct method Example

• Example 1
• ?CT(sample) CT (Target) - CT (Reference) DCt(ctr
  l SOCS3) 27-20 7
• DCt(tr SOCS3) 24-20 4
• ? CT ? CT (Sample) - ? CT (Calibrator) DDCt 4
  7 -3
• RQ 23 8
• Note Also Ct(tr SOCS3) Ct(ctrl SOCS3)
  27-24 3 because
• starting conc was equal (equal 18S)
• ? SOCS3 expression in treated sample is 8 times
  higher than in control sample.

ctrl 18S
ctrl SOCS3
ctrl SOCS3
tr 18S
ctrl 18S
tr 18S
tr SOCS3
tr SOCS3
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??Ct method Example

• Example 2
• ?CT(sample) CT (Target) - CT (Reference) DCt(ctr
  l SOCS3) 28-16 12
• DCt(tr SOCS3) 25-13 12
• ? CT ? CT (Sample) - ? CT (Calibrator) DDCt
  12 12 0
• RQ 20 1
• ? no difference in SOCS3 expression in
  treated and control sample!

ctrl 18S
ctrl SOCS3
ctrl SOCS3
tr 18S
ctrl 18S
tr 18S
tr SOCS3
tr SOCS3
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??Ct method

• no need for dilution series ? less material
  needed, faster
• BUT amplification efficiency of target and
  endogenous control must be comparable

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Efficiency of amplification
??Ct method
Changes in efficiency change the slope when you
use the ? logarithmic scale.
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Validation of efficiency

• equal efficiency or equal slopes for target and
• endogenous control
• - Acceptible slope 3.2 - 3.8 (Efficiency 83
  105 )

Target
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Target
y - 4. 586x 24.889 Effic 67
Endogenous control
y - 3.3683x 36.009 Effic 98
Value
t
C
D
y - 3.3276x 27.712 Effic 100
0
2
4
6
8
1
0
Log Input mRNA
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??Ct method

• Does the target have a similar amplification
  efficiency to the endogenous control?

YES
NO
??Ct method
Standard Curves
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Primers and probe design
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Primers and probe design

• Probe
• Tm 10ºC higher than Primer Tm (7ºC for Allelic
  Discrimination)
• 20 - 80 GC
• Length 9 - 40 bases
• No G on the 5end
• lt4 contiguous Gs
• Must not have more Gs than Cs

• Amplicon
• 50 - 150 bp in length
• As close to the probe as possible without
  overlapping

• Primer
• Tm 58 - 60ºC
• 20 - 80 GC
• Length 9 - 40
• lt2ºC difference in Tm between the two primers
• Maximum of 2 G or C
• at 3 end

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Primers and probe design

• Theoretical Tms may not always be accurate
• This would lead to an imbalance between the two
  primers
• ? Primer optimisation

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Primers and probe design
Primer Concentration Optimization MATRIX
FORWARD REVERSE
50nM
300nM
900nM
50nM
50/50
300/50
900/50
300nM
50/300
300/300
900/300
900nM
50/900
300/900
900/900
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Primer Optimisation for SYBR Green I

• Perform 50/300/900nM primer matrix
• Choose the optimal primer concentration
• Lowest Ct
• Highest Rn
• No amplification in negative control

Probe Optimisation (Taqman)

• Increase probe concentration from 50nM to 300nM
• Lowest Ct without excess probe

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Multiplex reactions

• Primers and probes for both target gene (SOCS3)
  and reference gene (18S) in the same tube
• Primers for reference gene (18S) must be limited

target gene
18 S
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APPLICATIONS

• Real time detection
• Quantitation of gene expression
• Quantitation of RNA, DNA, cDNA
• Viral quantitation
• Endpoint detection
• Allelic discrimination (SNP genotyping)
• Plus/minus studies
• Pathogen detection

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ALLELIC DISCRIMINATION
End point detection
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ALLELIC DISCRIMINATION
Principle 2 primers, 2 probes
FAM-labelled probe is specific for Allele
1 VIC-labelled probe is specific for Allele 2
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ALLELIC DISCRIMINATION
Mechanism

• relies on competition between the two probes
• Tm of the mismatched probe lt Tm of perfectly
  matched probe

Tamra
VIC
Tamra
FAM
?
Allele 1
?
Allele 1
Incorrect Probe
Correct Probe
Tm 55ºC
Tm 65ºC
Annealing/extension temperature of 60C allows
binding and cleavage of correct probe and
destabilisation of incorrect probe
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ALLELIC DISCRIMINATION
Typical output
FAM
VIC
Allele 1
Allele 2
VIC
FAM
? homozygote for allele 1
? homozygote for allele 2
FAM
VIC
Allele 12
? heterozygote
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ALLELIC DISCRIMINATION
Typical output
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Practical tips

• Prevention of contamination (gloves, filtered
  tips, pre- and post-PCR area)
• Precise pipetting
• Use triplicates
• Include positive and negative controls
• For gene expression quant. use intron-spanning
  primers to avoid genomic contamination or use
  DNAse treatment after RNA purification

genomic DNA
EXON 1
EXON 2
cDNA
EXON 1
EXON 2