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Real Time PCR
Mandeep Kaur
CONTENTS
 Real Time PCR
 Working principle
 Instruments used in RT-PCR
 Chemistries used inReal TimePCR
 Absolute and relative quantification
 Melting curve
 Precautions
 Applications
What is Real - Time PCR?
The Polymerase Chain Reaction (PCR) is a process for the
amplification of specific fragments of DNA.
Real-Time PCR - Modification of conventional PCR where product
accumulation is measured/ visualized “in real time” as the reaction
progresses, after each PCR cycle.
Real Time PCR is a simple method to determine amount of target
sequence or gene that is present in sample.
• Kary Banks Mullis was an American biochemist.
• In recognition of his invention of PCR, he shared the 1993 Nobel prize
in chemistry with Michel Smith.
• Roche molecular systems, Inc scientist, upgraded it to qPCRǃ
NATURE BIOTECHNOLOGY
History of Real Time PCR
 Higuchi et al (1993) recognised that the process of PCR could be
monitored by addition of a flourescent label that binds to the accumulating
PCR product. As the concentration of PCR product increases , the intensity
of the fluorescent signal also increases.
 They used EtBr (Ethidium Bromide) for the detection. This discovery
paved way for modern quantitative real time PCR (qPCR). When EtBr is
bound to double stranded DNA and excited by UV light, it fluoresces.
 1996 - TaqMan detection methods used, instead of EtBr, for real time
detection of PCR.
Traditional PCR (semiquantitative)
Gel
Electrophoresis
1
2
4
8
2n
n cycles
Principle of qRT PCR
• Monitors the progress of the PCR as it occurs (in “real time”) by reading
fluorescence intensities after each cycle.
• Intensities are proportionalto the numberof amplicons generated.
• More starting material, sooner an increase in fluorescence).
• Baseline= Basal level offluorescencedefinedduringtheinitial cyclesofPCR.
• Threshold= Fixed fluorescencelevel set above thebaseline.
• Rn = normalized Reporter signal, level of fluorescence detected during PCR. Calculated by dividing
probereporter dyesignalbypassivereferencesignal (ROX).
• Ct = thresholdCycle,PCR cycleat whichan increasein reporter fluorescenceabove abaseline signal
is firstdetected (cyclewhenfluorescencecrossesthe threshold).
Definitions
Ct is the cycle number at which the fluorescence passes the threshold
Ct Ct Ct Ct
Threshold
Baseline
Measuring Quantities
DNAamount≈ 2 Cycle Number
4 units
Ct=23
1 unit
Ct=25
1/8 unit
Ct=28
• There is relationship
between the starting
amount of DNA and
Ct value.
• More the initial
concentration, less
Ct value.
Instruments used in qRT-PCR
Real-time PCR instruments consistof THREE main
components:
1. Thermal Cycler (PCR machine)
2. Optical Module (to detect fluorescence inthe
tubes duringthe run)
3. Computer (to translate the fluorescence data
intomeaningfulresults)
The real-time software converts the fluorescent
signals ineach well to meaningful data.
How do we label PCR products?
SYBR® Green dyeTaqMan® probe
.
SYBR green dye
Intercalating dye.
Emits fluorescence only when binds to ds DNA.
More sensitive than EtBr.
SYBR®Green Chemistry
Advantages:
Easy to produce
Cheap, does not require probe design.
Easy to handle & safer than EtBr.
Limitations:
Detection of non-specific ds reaction products (binds
to irrespective of DNA sequence).
 Increased background.
Inhibition of PCR reaction
.
Melt Curve or Dissociation Curve
Target amplicon
Tm = temperature when 50% dissociated
Dissociation Curve
Dissociation Curve
Example: Presence of Primer Dimers
.
Taqman probe
Two primers + 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.
Taqman probe
Taqman probe
Taqman probe
Taqman probe
Cleavage (5’ nuclease activity of taq DNA polymerase).
• Increase of reporter signal proportional to amount of amplicon produced.
• Removes probe from target strand.
Mechanism:
Fluorescence Resonance Energy Transfer (FRET)
Taqman probe
Emission Profiles of VariousFluorophores
Fam Vic/
Joe Tamra
Rox
Multiplex reactions possible
• Reporters: FAM,TET,VIC,JOE
• Quenchers: TAMRA, MGB
• Passive reference: ROX
.
qRT-PCR displays four stages
• Linear ground phase
• Early exponential phase
• Linear exponential phase (log phase)
• Plateau phase
 Linear ground phase: In the first phase, PCR is just starting, fluorescent signal
has not risen above background.
 Early exponential phase: The second phase is where fluorescent signal just
rise significantly above the background, the cycle at which occurs is called
cycle threshold (Ct).
 Linear exponential phase (log phase): In linear exponential phase, PCR is in
its optimal amplification stage with doubling PCR products in every cycle.
 Plateau phase: The last phase is when substrates are exhausted and Taq DNA
polymerase is in its end of life, fluorescent signal will no longer increase.
Quantification of gene expression
by qRT-PCR
 There are two basic types of qRT-PCR analysis:
 Absolute quantification
 Relative quantification
 The choice is based on both the experimental goals and available
resources.
 Absolute quantification: It determines the expression levels in absolute
number of copies, using a calibration curve.
 Relative quantification: It determines the fold changes in expression between
two samples.
Absolute Quantification
• Data fromRT PCR are CT or threshold cycle values.
• CT: Cycle number at whichdetectable signal isachieved.
• Greater Initial Concentration Less Ct Value .
• It compares the expression relative to a house keeping gene
and/or to another reference sample.
• The comparative Ct method compares the expression levels
between two samples, for example Gene Of Interest (GOI) in
sample A vs sample B.
..
Relative quantification
.
Research article
.
qRT-PCR analysis
 Total RNA of grape leaves was extracted using an RNAprep Pure Plant Kit.
 RNA quality and concentration were detected through agarose gel
electrophoresis and spectrophotometry
 The complementary DNA was synthesized using HiScript® II Reverse
Transcriptase and then subjected to qRT-PCR on an Opticon
thermocycler using SYBR Green PCR master mix.
 VviActin (accession: EC969944) was used as the reference gene, and
specific primer pairs for relevant genes were designed using Primer-
BLAST. The specificity of designed primers was verified through gel
electrophoresis.
 The PCR reactions were set under the following conditions: 95 °C for 10
min, 40 cycles of 95 °C for 10 s and 60 °C for 30 s.
 Each sample was analyzed with three biological and technical replicates.
 The relative expression levels of the target genes were examined using the
2 −ΔΔCt method.
 Significant difference was determined by t-test using the R program.
Advantages of Real-Time PCR
Collects data in the EXPONENTIAL GROWTH PHASE.
REAL TIME: permanent record of amplification.
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.
.
Applications of Real Time PCR
.
• Gene expression analysis.
• Detection of mutant genes.
• Gene copy number measurement.
• Pathogen/microbe detection and
quantification or viral load estimation.
• Single nucleotide polymorphism analysis
.................so many other applications
Real time cyclers available in the
market
• LightCycler, Roche Diagnostics.
• ABI Prism 7000.
• iCycler Iq, Bio Rad.
• Stratagene Mx3000P, Agilent Technologies.
• Smart Cycler, Cepheid.
• Rotor Gene, Corbett Research.
• Techne Quantica, GMI Inc.
THANK
YOU

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qRT PCR

  • 2. CONTENTS  Real Time PCR  Working principle  Instruments used in RT-PCR  Chemistries used inReal TimePCR  Absolute and relative quantification  Melting curve  Precautions  Applications
  • 3. What is Real - Time PCR? The Polymerase Chain Reaction (PCR) is a process for the amplification of specific fragments of DNA. Real-Time PCR - Modification of conventional PCR where product accumulation is measured/ visualized “in real time” as the reaction progresses, after each PCR cycle. Real Time PCR is a simple method to determine amount of target sequence or gene that is present in sample.
  • 4. • Kary Banks Mullis was an American biochemist. • In recognition of his invention of PCR, he shared the 1993 Nobel prize in chemistry with Michel Smith. • Roche molecular systems, Inc scientist, upgraded it to qPCRǃ
  • 6. History of Real Time PCR  Higuchi et al (1993) recognised that the process of PCR could be monitored by addition of a flourescent label that binds to the accumulating PCR product. As the concentration of PCR product increases , the intensity of the fluorescent signal also increases.  They used EtBr (Ethidium Bromide) for the detection. This discovery paved way for modern quantitative real time PCR (qPCR). When EtBr is bound to double stranded DNA and excited by UV light, it fluoresces.  1996 - TaqMan detection methods used, instead of EtBr, for real time detection of PCR.
  • 8. Principle of qRT PCR • Monitors the progress of the PCR as it occurs (in “real time”) by reading fluorescence intensities after each cycle. • Intensities are proportionalto the numberof amplicons generated. • More starting material, sooner an increase in fluorescence).
  • 9. • Baseline= Basal level offluorescencedefinedduringtheinitial cyclesofPCR. • Threshold= Fixed fluorescencelevel set above thebaseline. • Rn = normalized Reporter signal, level of fluorescence detected during PCR. Calculated by dividing probereporter dyesignalbypassivereferencesignal (ROX). • Ct = thresholdCycle,PCR cycleat whichan increasein reporter fluorescenceabove abaseline signal is firstdetected (cyclewhenfluorescencecrossesthe threshold). Definitions
  • 10. Ct is the cycle number at which the fluorescence passes the threshold Ct Ct Ct Ct Threshold Baseline
  • 11. Measuring Quantities DNAamount≈ 2 Cycle Number 4 units Ct=23 1 unit Ct=25 1/8 unit Ct=28 • There is relationship between the starting amount of DNA and Ct value. • More the initial concentration, less Ct value.
  • 12. Instruments used in qRT-PCR Real-time PCR instruments consistof THREE main components: 1. Thermal Cycler (PCR machine) 2. Optical Module (to detect fluorescence inthe tubes duringthe run) 3. Computer (to translate the fluorescence data intomeaningfulresults) The real-time software converts the fluorescent signals ineach well to meaningful data.
  • 13.
  • 14. How do we label PCR products? SYBR® Green dyeTaqMan® probe
  • 15. . SYBR green dye Intercalating dye. Emits fluorescence only when binds to ds DNA. More sensitive than EtBr.
  • 17. Advantages: Easy to produce Cheap, does not require probe design. Easy to handle & safer than EtBr. Limitations: Detection of non-specific ds reaction products (binds to irrespective of DNA sequence).  Increased background. Inhibition of PCR reaction
  • 18. . Melt Curve or Dissociation Curve
  • 19. Target amplicon Tm = temperature when 50% dissociated Dissociation Curve
  • 21. . Taqman probe Two primers + 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.
  • 24.
  • 27. Cleavage (5’ nuclease activity of taq DNA polymerase). • Increase of reporter signal proportional to amount of amplicon produced. • Removes probe from target strand. Mechanism: Fluorescence Resonance Energy Transfer (FRET)
  • 29. Emission Profiles of VariousFluorophores Fam Vic/ Joe Tamra Rox Multiplex reactions possible • Reporters: FAM,TET,VIC,JOE • Quenchers: TAMRA, MGB • Passive reference: ROX
  • 30. . qRT-PCR displays four stages • Linear ground phase • Early exponential phase • Linear exponential phase (log phase) • Plateau phase
  • 31.  Linear ground phase: In the first phase, PCR is just starting, fluorescent signal has not risen above background.  Early exponential phase: The second phase is where fluorescent signal just rise significantly above the background, the cycle at which occurs is called cycle threshold (Ct).
  • 32.  Linear exponential phase (log phase): In linear exponential phase, PCR is in its optimal amplification stage with doubling PCR products in every cycle.  Plateau phase: The last phase is when substrates are exhausted and Taq DNA polymerase is in its end of life, fluorescent signal will no longer increase.
  • 33. Quantification of gene expression by qRT-PCR  There are two basic types of qRT-PCR analysis:  Absolute quantification  Relative quantification  The choice is based on both the experimental goals and available resources.  Absolute quantification: It determines the expression levels in absolute number of copies, using a calibration curve.  Relative quantification: It determines the fold changes in expression between two samples.
  • 34. Absolute Quantification • Data fromRT PCR are CT or threshold cycle values. • CT: Cycle number at whichdetectable signal isachieved. • Greater Initial Concentration Less Ct Value .
  • 35. • It compares the expression relative to a house keeping gene and/or to another reference sample. • The comparative Ct method compares the expression levels between two samples, for example Gene Of Interest (GOI) in sample A vs sample B. .. Relative quantification .
  • 36.
  • 38.
  • 39.
  • 40.
  • 41. . qRT-PCR analysis  Total RNA of grape leaves was extracted using an RNAprep Pure Plant Kit.  RNA quality and concentration were detected through agarose gel electrophoresis and spectrophotometry  The complementary DNA was synthesized using HiScript® II Reverse Transcriptase and then subjected to qRT-PCR on an Opticon thermocycler using SYBR Green PCR master mix.  VviActin (accession: EC969944) was used as the reference gene, and specific primer pairs for relevant genes were designed using Primer- BLAST. The specificity of designed primers was verified through gel electrophoresis.  The PCR reactions were set under the following conditions: 95 °C for 10 min, 40 cycles of 95 °C for 10 s and 60 °C for 30 s.  Each sample was analyzed with three biological and technical replicates.  The relative expression levels of the target genes were examined using the 2 −ΔΔCt method.  Significant difference was determined by t-test using the R program.
  • 42.
  • 43.
  • 44.
  • 45. Advantages of Real-Time PCR Collects data in the EXPONENTIAL GROWTH PHASE. REAL TIME: permanent record of amplification. 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.
  • 46. . Applications of Real Time PCR . • Gene expression analysis. • Detection of mutant genes. • Gene copy number measurement. • Pathogen/microbe detection and quantification or viral load estimation. • Single nucleotide polymorphism analysis .................so many other applications
  • 47. Real time cyclers available in the market • LightCycler, Roche Diagnostics. • ABI Prism 7000. • iCycler Iq, Bio Rad. • Stratagene Mx3000P, Agilent Technologies. • Smart Cycler, Cepheid. • Rotor Gene, Corbett Research. • Techne Quantica, GMI Inc.