This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
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Introduction to real-Time Quantitative PCR (qPCR) - Download the slides
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Introduction To Real-Time Quantitative PCR (qPCR)
Dr. Vishwadeepak Tripathi, Global Marketing Manager – QIAGEN
1Introduction To Real-Time Quantitative PCR (qPCR)
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diagnosis, prevention or treatment of a disease.
For up-to-date licensing information and product-specific
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2Introduction To Real-Time Quantitative PCR (qPCR)
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How does qPCR work?
Finish
Line
Car 2
Car 1
Question: How far apart are the 2 cars?
Cars race at same speed to finish line
As car 1 crosses finish line, calculate time for car 2 to finish
Calculate difference in starting position mathematically (d = rate x time)
Introduction To Real-Time Quantitative PCR (qPCR) 3
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How does qPCR work?
Finish
Line
Car 2
Car 1
Question: How far apart are the 2 cars?
Many cars; how to differentiate cars of interest
Introduction To Real-Time Quantitative PCR (qPCR) 4
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Agenda
Introduction To Real-Time Quantitative PCR (qPCR) 5
What is qPCR?
Factors for successful qPCR
Reporter Technologies
Characteristics of a good qPCR Assay
Analyzing qPCR Curves
Data & Analysis
Q&A
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Agenda
Introduction To Real-Time Quantitative PCR (qPCR) 6
What is qPCR?
Applications and Example
Factors for successful qPCR
Reporter Technologies
Characteristics of a good qPCR Assay
Analyzing qPCR Curves
Data & Analysis
Q&A
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What is qPCR? Applications and Example
What does Real-Time qPCR stand for?
Quantitative Polymerase Chain Reaction (qPCR) is a sensitive and reliable method for
detection and quantification of nucleic acid (DNA & RNA) levels.
It is based on detection and quantification of fluorescence emitted from a reporter molecule
in real time.
This detection occurs during the accumulation of the PCR product with each cycle of
amplification
Monitor the PCR reaction during early & exponential phase, where the first significant
increase in the amount of PCR product correlates to the initial amount of target template.
Introduction To Real-Time Quantitative PCR (qPCR) 7
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What is qPCR? Applications and Example
Applications for qPCR
RNA
DNA
• Gene Expression Profiling Analysis
• miRNA Expression Profiling Analysis
• SNP Genotyping & allelic discrimination
• Somatic Mutation Analysis
• Copy Number Detection/Variation Analysis
• Chromatin IP Quantification
• DNA Methylation Detection
• Pathogen Detection
• Viral Quantification
Introduction To Real-Time Quantitative PCR (qPCR) 8
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What is qPCR? Applications and Example
qPCR Work Flow: A Brief Look
Sample
input
Sample
QC
• DNA
• RNA (total, mRNA,
miRNA)
• qPCR Assay:
SYBR
Green/Probe
Assay Design
Assay
Optimization
Instrument
Set up &
thermal
cycling
cDNA
Synthesis
Real Time
PCR Set Up
Data Output
& Analysis
Introduction To Real-Time Quantitative PCR (qPCR) 9
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What is qPCR? Applications and Example
Applications for qPCR
RNA
DNA
• Gene Expression Profiling Analysis
• miRNA Expression Profiling Analysis
• SNP Genotyping & allelic discrimination
• Somatic Mutation Analysis
• Copy Number Detection/Variation Analysis
• Chromatin IP Quantification
• DNA Methylation Detection
• Pathogen Detection
• Viral Quantification
Introduction To Real-Time Quantitative PCR (qPCR) 10
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What is qPCR? Applications and Example
qPCR for gene expression: application example
Gene expression changes during
differentiation
Differentiation protocol
Collect Total RNA at different time
points (miRNeasy Mini Kit)
Measure 1 HKG and 1 GOI (TNFa)
Repeat experiment 3x (biological
replicates)
Introduction To Real-Time Quantitative PCR (qPCR) 11
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What is qPCR? Applications and Example
Work Flow: Gene expression profiling
Total RNA
Sample
QC
• qPCR Assay:
SYBR Green
Assay Design
Assay
Optimization
Thermo-
cycling
Real Time
PCR Set Up
Data
Analysis
cDNA
Synthesis
Introduction To Real-Time Quantitative PCR (qPCR) 12
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Factors Critical for successful qPCR
Components of Successful qPCR experiment
Introduction To Real-Time Quantitative PCR (qPCR) 13
Template quality - DNA or RNA sample preparation
• Appropriate sample prep kits/reagents
• Inhibitors can compromise RT or PCR
Reverse transcription - convert RNA to cDNA
• type of RT
• type of primers
• Controls
Assay design - chemistry, specificity, PCR efficiency, throughput & cost
• Choose validated assay, or need to validate our own?
Running PCR
• Commercial mastermix or make own (primer, probe, master mix)
Data analysis tool
• User friendly
• Streamlined data analysis module
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Agenda
Introduction To Real-Time Quantitative PCR (qPCR) 14
What is qPCR?
Factors for successful qPCR
RNA Quality and Integrity
Reverse Transcription
Reporter Technologies
Characteristics of a good qPCR Assay
Analyzing qPCR Curves
Data & Analysis
Q&A
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RNA Quality and Integrity
RNA Isolation
Title only (a)
Considerations
• Sample type/amount
• Target (total RNA, mRNA, miRNA)
• Throughput
• Difficult samples (stool, FFPE)
Challenges
• Contamination (gDNA)
• Yield
• Quality
Method
• Qiazol/Trizol?
• Column based method (RNeasy)?
• Both?
◦ Efficient lysis and inhibition of RNases;
molecular grade RNADonec quam felis,
ultricies nec
• miRNA? Use a kit specific for miRNA and mRNA
Introduction To Real-Time Quantitative PCR (qPCR) 15
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RNA Quality and Integrity
Integrity:
Purity/ Quantity:
Spectrophotometer: measure 260/280 and 260/230
• OD260 is used to calculate amount of nucleic
acid
◦ 260/280 ratio
typical minimum value 1.8-2.0
◦ 260/230 ratio
– typical minimum value 1.7
• Low ratio may indicate a contaminant: protein,
QIAzol, Carbohydrates, Glycogen
Denaturing RNA Agarose Gel
• Usually through ribosomal bands
QIAxcel/ Bioanalyzer
• Capillary electrophoresis
• Automate RNA integrity analysis
• RNA integrity analysis number
Introduction To Real-Time Quantitative PCR (qPCR) 16
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Agenda
Introduction To Real-Time Quantitative PCR (qPCR) 17
What is qPCR?
Factors for successful qPCR
RNA Quality and Integrity
Reverse Transcription
Reporter Technologies
Characteristics of a good qPCR Assay
Analyzing qPCR Curves
Data & Analysis
Q&A
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Reverse Transcription
Reverse Transcription: Used to make cDNA copy of RNA
Important Notes
Reagents
Introduction To Real-Time Quantitative PCR (qPCR) 18
• Reverse transcriptase – many different kinds
• dNTPs
• Buffers for RT
• Primers
◦ Random pentamers or hexamers
◦ Oligo-dT
◦ Both
• Controls
• RT reaction is linear
• Do not try to reverse transcribe too much RNA
• Sensitivity of qPCR step is dependent on good
RT reaction
• Monitor RT reaction for equal efficiency across
samples
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Factors Critical For A Successful qPCR Assay
qPCR Components
A. Templates:
RNA
Starting amount:
~10-1000 copies of NA per qPCR assay
For a low-expressed gene, need 10ng equivalent
of RNA per reaction
Start with about 100pg to 1ug RNA
Reverse Transcription
One-Step PCR - 1 tube reaction
Two-Step PCR - 2 separate reactions
B. Primers/Probes
C. Master Mix
DNA polymerase
Mg++
dNTPs
Buffer
Passive reference dye
D. Cycling Conditions
Denature>Annealing>Extension
Denature>Annealing/Extension
Introduction To Real-Time Quantitative PCR (qPCR) 19
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Agenda
Introduction To Real-Time Quantitative PCR (qPCR) 20
What is qPCR?
Factors for successful qPCR
Reporter Technologies
qPCR in Action
Characteristics of a good qPCR Assay
Analyzing qPCR Curves
Data & Analysis
Q&A
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qPCR in Action
DNA Template
(ss or ds)
Polymerase
dNTPs
Primers (2)
PCR= Polymerase Chain Reaction
Exponential Amplification of DNA in single tube
All reagents in excess (non-limiting)
What is in a PCR Reaction?
Components
Thermostable polymerase
dNTPs
Primers
Template
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qPCR in Action
Polymerase
dNTPs
Primers (2)
1. Heat denature template (~95C)
2. Annealing (~60C)
3. Extension (~60C)
4. Repeat (~95C)
DNA Template
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qPCR in Action
DNA Template
Heat denature
Polymerase
dNTPs
Primers (2)
1. Heat denature template (~95C)
2. Annealing (~60C)
3. Extension (~60C)
4. Repeat (~95C)
Introduction To Real-Time Quantitative PCR (qPCR) 23
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qPCR in Action
Polymerase
dNTPs
Primers (2)
1. Heat denature template (~95C)
2. Annealing (~60C)
3. Extension (~60C)
4. Repeat (~95C)
DNA Template
Introduction To Real-Time Quantitative PCR (qPCR) 24
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qPCR in Action
DNA Template
(ss or ds)
Polymerase
Polymerase
dNTPs
Primers (2)
Polymerase
1. Heat denature template (~95C)
2. Annealing (~60C)
3. Extension (~60C)
4. Repeat (~95C)
Introduction To Real-Time Quantitative PCR (qPCR) 25
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qPCR in Action
DNA Template
(ss or ds)
Polymerase
dNTPs
Primers (2)
1. Heat denature template (~95C)
2. Annealing (~60C)
3. Extension (~60C)
4. Repeat (~95C)
Polymerase
Polymerase
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qPCR in Action
DNA Template
(ss or ds)
Polymerase
Polymerase
Polymerase
dNTPs
Primers (2)
1. Heat denature template (~95C)
2. Annealing (~60C)
3. Extension (~60C)
4. Repeat (~95C)
Introduction To Real-Time Quantitative PCR (qPCR) 27
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qPCR in Action
DNA Template
(ss or ds)
Polymerase
dNTPs
Primers (2)
1. Heat denature template (~95C)
2. Annealing (~60C)
3. Extension (~60C)
4. Repeat (~95C)
Introduction To Real-Time Quantitative PCR (qPCR) 28
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qPCR in Action
1. Heat denature template (~95C)
2. Annealing (~60C)
3. Extension (~60C)
4. Measure amount of PCR Product
5. Repeat (~95C)
Polymerase
dNTPs
Primers (2)
DNA Template
(ss or ds)
How do you make this into quantitative PCR?
Measure DNA amount at end of each cycle to get
ratio of DNA or absolute amount (if using a
standard)
Introduction To Real-Time Quantitative PCR (qPCR) 29
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Reporter chemistries
DNA binding agents Hydrolysis Probes
SYBR® I Dye Dual-labeled Hydrolysis
(Taqman®) probe
Introduction To Real-Time Quantitative PCR (qPCR) 30
Others, such as hybridization
probes
Molecular beacon and
scorpion probes
Real-Time qPCR Fluorescence Chemistry
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Reporter chemistries: SYBR® Green I Assay
Fluorescent SYBR I
SYBR I binds to double-strand DNA but not single
strand DNA. Little fluorescence emitted from SYBR
I in solution.
SYBR I upon binding to double-strand DNA emits
fluorescence very brightly
The SYBR I signal intensities correlate with DNA
amplified (amplicon amount) thus the initial sample
input amounts
Introduction To Real-Time Quantitative PCR (qPCR) 31
Simple & cost saving; high specificity is required since SYBR I binds all double-
strand DNA (non-specific or primer dimer)
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Reporter chemistries: Hydrolysis Probe Assay
The fluorescence of the reporter dye is suppressed
by the quencher
Primer binding followed by extension
Probe cleavage by Taq to free the reporter dye thus
the fluorescence intensity correlates with the initial
sample input amounts. Taq has 5’→3’ exonuclease
activity
Introduction To Real-Time Quantitative PCR (qPCR) 32
Each amplicon needs a sequence-specific probe; increased cost & time
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Reporter Chemistries: Understanding Kinetics in PCR
Amplification Plot (Linear scale)
Exponential Phase
Plateau phase
• End-point PCR data collection at plateau (gel
analysis)
• Reactions varying due to reagent depletion &
decreased PCR efficiencies (enzyme activity,
more product competing for primer annealing)
• Real time PCR does early phase detection
• proportional to input amounts
• 2n=dilution factor
Introduction To Real-Time Quantitative PCR (qPCR) 33
Amplification Plot (Linear scale)
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Reporter Chemistries: Understanding Kinetics in PCR
Exponential Phase
Plateau phase
• End-point PCR data collection at plateau (gel
analysis)
• Reactions varying due to reagent depletion &
decreased PCR efficiencies (enzyme activity,
more product competing for primer annealing)
• Real time PCR does early phase detection
• proportional to input amounts
• 2n=dilution factor
Introduction To Real-Time Quantitative PCR (qPCR) 34
Plateau
107 106 105
FluorescenceSignal
Amplification Plot (Linear scale)
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Agenda
Introduction To Real-Time Quantitative PCR (qPCR) 35
What is qPCR?
Factors for successful qPCR
Reporter Technologies
Characteristics of a good qPCR Assay
Analyzing qPCR Curves
Data & Analysis
Q&A
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Characteristics of a good qPCR Assay
What factors do you need to address to create a good PCR Assay?
Amplification efficiency
100% during exponential phase (template product doubles with each cycle)
Sensitivity
Able to detect down to reasonable quantities of template in 1 reaction (10-50 copies)
Specificity
1 assay, 1 target: (no off-target amplification or primer-dimers)
Melt-curve analysis - 1 peak, 1 product
Agarose gel
Dynamic Range
Ability to detect genes with varied expression levels
Reproducibility
Confidence in your results, enables profiling of multiple genes in the same sample
All lab members get the same results
Technical reproducibility ensures changes seen in results are due to the biology and not the
technology or sample handling
Introduction To Real-Time Quantitative PCR (qPCR) 36
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Characteristics of a good qPCR Assay: Amplification Efficiency
Single curve analysis
Standard curve
• X axis – dilution
• Y axis - Ct value
• Amp efficiency = 10(-1/slope) -1 *100
• PCR Miner:
http://miner.ewindup.info/version2
• “DART”: www.gene-
quantification.de/DART_PCR_version_1.0
.xls
Introduction To Real-Time Quantitative PCR (qPCR) 37
Amplification Efficiency: reliable and accurate experiment (Two Methods)
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Characteristics of a good qPCR Assay: Sensitivity
Two Methods:
Sensitivity
How many copies can my assay detect?
• Important for low expressed genes or
where there is limited sample
• Method 1: Use primers to make PCR
product, T/A clone, grow-up, isolate,
quantitate and use for qPCR reactions
• Method 2: Use gDNA as template and use
mass of gDNA to calculate copy number
and assume 1 target per genome (or
actually calculate targets using
bioinformatics)
Introduction To Real-Time Quantitative PCR (qPCR) 38
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Characteristics of a good qPCR Assay: Amplification Efficiency
Introduction To Real-Time Quantitative PCR (qPCR) 39
Specificity:
1 target amplified
Two Methods:
Melt Curve analysis
• 1 peak, 1 product
Agarose gel
• Band at correct size
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Characteristics of a good qPCR Assay: Specificity
Plot - Normalized Reporter
Melting Curve Analysis
Gene A Tm: 77.36 Gene B Tm: 78.94
NormalizedFluorescenceSignal
Temperature
50% fluorescence
drop
Rn
The General Program Steps
Heat to 94°C to denature DNA
Cooling to 60°C to let DNA
double strands anneal
Slowly heat (increase temp. to
0.2°C/sec) while plotting the
fluorescent signal vs.
temperature.
As the temp increases, DNA
melts, fluorescent signal should
decrease.
Significant drop in signal when
50% DNA melts.
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Characteristics of a good qPCR Assay: Specificity
Plot -1st negative Derivative Reporter
Temperature
-deltaF/deltaT(thechangerate)
Gene A Tm: 77.36
Gene B Tm: 78.94
Single melt curve of each
amplicon is required for specificity
validation
Melting Curve Analysis
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Agenda
Introduction To Real-Time Quantitative PCR (qPCR) 42
What is qPCR?
Factors for successful qPCR
Reporter Technologies
Characteristics of a good qPCR Assay
Analyzing qPCR Curves
Data & Analysis
Q&A
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Analyzing qPCR curves: How to Define Baseline
Manual Baseline Option
Automated Baseline Option
If an instrument has an adaptive baseline
function
Use linear view of the plot
• Set up the baseline reading from cycle #2 to the
cycle that is 2 cycles before the earliest visible
amplification
• Usually a baseline falls in 3-15 cycles
Introduction To Real-Time Quantitative PCR (qPCR) 43
Linear Amplification Plot
Baseline
Ct
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Analyzing qPCR curves: How to Define Thershold
Define the Threshold
Use log view of amplification plot
• Threshold should be higher than baseline (higher
than the noise level)
• Threshold should at LOWER 1/3 or 1/2 of the
linear phase of amplification
• Linear phase = exponential phase
• Different runs across samples for the same
experiments should have the same threshold for
comparison
Introduction To Real-Time Quantitative PCR (qPCR) 44
Log View Amplification Plot
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Agenda
Introduction To Real-Time Quantitative PCR (qPCR) 45
What is qPCR?
Factors for successful qPCR
Reporter Technologies
Characteristics of a good qPCR Assay
Analyzing qPCR Curves
Data & Analysis
Q&A
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Data Analysis: Biological replicates and technical replicates
Replicates
Introduction To Real-Time Quantitative PCR (qPCR) 46
Technical Replicates
• Look for variation due to technique, reagents, equipment...
• RT2 PCR Arrays are highly reproducible
◦ Triplicate RTC and PPC show technical reproducibility on each plate, and comparable results across
plates
Biological Replicates
• Need at least 3 for p-value
• Look for variation due to biology
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Data Analysis: Housekeeping/Reference Genes
GOI in control cells GOI in treated cells
Ref Gene in control cells Ref Gene in drug treated cells
Any changes?
Reference gene
Expression level remains consistent under experimental conditions/different tissues
Aimed to normalize possible variations during:
Sample prep & handling (e.g. use the same number of cells from a start)
RNA isolation (RNA quality and quantity)
Reverse transcription efficiency across samples/experiments
PCR reaction set up
PCR reaction amplification efficiencies
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Data Analysis: Commonly Used Housekeeping Genes
Introduction To Real-Time Quantitative PCR (qPCR) 48
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Data & analysis
1. Average Ct values for all gene replicates
2. Calculate ΔCt value between GOI and HKG for each experiment
3. Average ΔCt values between experiments (replicates)
4. Calculate ΔΔCt values (ΔCt experiment- ΔCt control)
5. Calculate Fold Change 2(-ΔΔCt)
Introduction To Real-Time Quantitative PCR (qPCR) 49
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Data & analysis: Delta Delta Ct Method - Amplification Plots
Ref
GOI
GAPDH
TNFa
∆∆Ct = ∆Ct (TNFαtreat-GAPDHtreat) - ∆Ct (TNFαcontrol-GAPDHcontrol)
The fold change = 2(-∆∆Ct)
Ct Ct Ct Ct
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Data & analysis
1. Average Ct values for all gene replicates
2. Calculate Delta Ct value: GOI-HKG
3. Average Delta Ct values between experiments
4. Calculate Delta-Delta Ct values (Delta Ct experiment- Delta Ct control)
5. Calculate Fold Change 2(-Delta Delta Ct)
17.1 17.2 17.2 qPCR replicates
Introduction To Real-Time Quantitative PCR (qPCR) 52
TNFa is up-regulated 32 fold in the treated cells versus the control
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Data Analysis Tools
Introduction To Real-Time Quantitative PCR (qPCR) 54
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Introduction To Real-Time Quantitative PCR (qPCR) 55
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Agenda
Introduction To Real-Time Quantitative PCR (qPCR) 56
What is qPCR?
Factors for successful qPCR
Reporter Technologies
Characteristics of a good qPCR Assay
Analyzing qPCR Curves
Data & Analysis
Q&A
57. Sample to Insight
Thank you for attending!
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Questions?
Contact QIAGEN
qiawebinars@qiagen.com
Vishwadeepak Tripathi, PhD
Vishwadeepak.Tripathi@qiagen.com
More information is also available at:
www.qiagen.com
Introduction To Real-Time Quantitative PCR (qPCR)