Introduction To Real-Time Quantitative PCR (qPCR)

Dr. Vishwadeepak Tripathi, Global Marketing Manager – QIAGEN

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Introduction To Real-Time Quantitative PCR (qPCR) 1
 Legal disclaimer

QIAGEN products shown here are intended for molecular

biology applications. These products are not intended for the
diagnosis, prevention or treatment of a disease.

For up-to-date licensing information and product-specific

disclaimers, see the respective QIAGEN kit handbook or user
manual. QIAGEN kit handbooks and user manuals are available
at www.qiagen.com or can be requested from QIAGEN
Technical Services or your local distributor.

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Introduction To Real-Time Quantitative PCR (qPCR)
 How does qPCR work?

Finish
Car 2
Line

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)

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Introduction To Real-Time Quantitative PCR (qPCR)
 How does qPCR work?

Finish
Car 2
Line

Car 1

Question: How far apart are the 2 cars?

 Many cars; how to differentiate cars of interest

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Introduction To Real-Time Quantitative PCR (qPCR)
 Agenda

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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Introduction To Real-Time Quantitative PCR (qPCR)
 Agenda

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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Introduction To Real-Time Quantitative PCR (qPCR)
 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.

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Introduction To Real-Time Quantitative PCR (qPCR)
 What is qPCR? Applications and Example

Applications for qPCR

• Gene Expression Profiling Analysis

RNA
• miRNA Expression ProfilingAnalysis

• SNP Genotyping & allelic discrimination

• Somatic Mutation Analysis
• Copy Number Detection/Variation Analysis
DNA • Chromatin IP Quantification
• DNA Methylation Detection
• Pathogen Detection
• Viral Quantification
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Introduction To Real-Time Quantitative PCR (qPCR) 8

 What is qPCR? Applications and Example

qPCR Work Flow: A Brief Look

Instrument
Sample Sample cDNA Real Time Set up & Data Output
input QC Synthesis PCR Set Up thermal & Analysis
cycling

• DNA • qPCR Assay:

• RNA (total, mRNA, SYBR
miRNA) Green/Probe
Assay Design
Assay
Optimization

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Introduction To Real-Time Quantitative PCR (qPCR)
 What is qPCR? Applications and Example

Applications for qPCR

• Gene Expression Profiling Analysis

RNA
• miRNA Expression ProfilingAnalysis

• SNP Genotyping & allelic discrimination

• Somatic Mutation Analysis
• Copy Number Detection/Variation Analysis
DNA • Chromatin IP Quantification
• DNA Methylation Detection
• Pathogen Detection
• Viral Quantification
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Introduction To Real-Time Quantitative PCR (qPCR) 10

 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)
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Introduction To Real-Time Quantitative PCR (qPCR)
 What is qPCR? Applications and Example

Work Flow: Gene expression profiling

Sample cDNA Real Time Thermo- Data

Total RNA
QC Synthesis PCR Set Up cycling Analysis

• qPCR Assay: SYBR Green Assay Design

Assay
Optimization

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Introduction To Real-Time Quantitative PCR (qPCR)
 Factors Critical for successful qPCR

Components of Successful qPCR experiment

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, mastermix) Data analysis tool
• User friendly
• Streamlined data analysis module

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Introduction To Real-Time Quantitative PCR (qPCR)
 Agenda

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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Introduction To Real-Time Quantitative PCR (qPCR)
 Title only (a)

RNA Quality and Integrity

RNA Isolation

Considerations Method
• Sample type/amount • Qiazol/Trizol?
• Target (total RNA, mRNA, miRNA) • Column based method (RNeasy)?
• Throughput • Both?
•Difficult samples (stool, FFPE) ◦ Efficient lysis and inhibition of RNases;
Challenges molecular grade RNADonec quam felis, ultricies nec

• Contamination (gDNA) • miRNA? Use a kit specific for miRNA and mRNA
• Yield
• Quality

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Introduction To Real-Time Quantitative PCR (qPCR) 15
 RNA Quality and 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

Integrity:
Denaturing RNA AgaroseGel
• Usually through ribosomal bands

QIAxcel/ Bioanalyzer
• Capillary electrophoresis
• Automate RNA integrity analysis
• RNA integrity analysis number

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Introduction To Real-Time Quantitative PCR (qPCR)
 Agenda

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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Introduction To Real-Time Quantitative PCR (qPCR)
 Reverse Transcription

Reagents
• Reverse transcriptase – many different kinds
• dNTPs
• Buffers for RT
• Primers
◦ Random pentamers or hexamers
◦ Oligo-dT
◦ Both
• Controls

Important Notes
• 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 efficiencyacross samples

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Introduction To Real-Time Quantitative PCR (qPCR)
 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

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Introduction To Real-Time Quantitative PCR (qPCR) 19
 Agenda

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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Introduction To Real-Time Quantitative PCR (qPCR)
 qPCR in Action

DNATemplate
(ss or ds)

PCR= Polymerase Chain Reaction

Exponential Amplification of DNA in single
Polymerase
tube All reagents in excess (non-limiting)

What is in a PCR Reaction?

dNTPs
 Components
 Thermostable polymerase
 dNTPs Primers (2)
 Primers
 Template

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Introduction To Real-Time Quantitative PCR (qPCR)
 qPCR in Action

DNATemplate

Polymerase

dNTPs
1. Heat denature template (~95C)
2. Annealing (~60C)
Primers (2)
3. Extension (~60C)
4. Repeat (~95C)
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Introduction To Real-Time Quantitative PCR (qPCR)
 qPCR in Action

Heat denature
DNATemplate

Polymerase

dNTPs
1. Heat denature template (~95C)
2. Annealing (~60C)
Primers (2)
3. Extension (~60C)
4. Repeat (~95C)
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Introduction To Real-Time Quantitative PCR (qPCR)
 qPCR in Action

DNATemplate

Polymerase

dNTPs
1. Heat denature template (~95C)
2. Annealing (~60C)
Primers (2)
3. Extension (~60C)
4. Repeat (~95C)
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Introduction To Real-Time Quantitative PCR (qPCR)
 qPCR in Action

Polymerase

Polymerase DNATe
(ss or

Polymerase

dNTPs
1. Heat denature template (~95C)

2. Annealing (~60C)
Primers (2)
3. Extension (~60C)
 4. Repeat (~95C)
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Introduction To Real-Time Quantitative PCR (qPCR)
 qPCR in Action

Polymerase

Polymerase DNATemplate
(ss or ds)

Polymerase

dNTPs
1. Heat denature template (~95C)
2. Annealing (~60C)
Primers (2)
3. Extension (~60C)
4. Repeat (~95C)
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Introduction To Real-Time Quantitative PCR (qPCR)
 qPCR in Action

Polymerase

DNATemplate
(ss or ds)

Polymerase

Polymerase

dNTPs
1. Heat denature template (~95C)
2. Annealing (~60C)
Primers (2)
3. Extension (~60C)
4. Repeat (~95C)
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Introduction To Real-Time Quantitative PCR (qPCR)
 qPCR in Action

DNATemplate
(ss or ds)

Polymerase

dNTPs
1. Heat denature template (~95C)
2. Annealing (~60C)
Primers (2)
3. Extension (~60C)
4. Repeat (~95C)
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Introduction To Real-Time Quantitative PCR (qPCR)
 qPCR in Action

DNATemplate
(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) Polymerase

2. Heat denature template (~95C)

3. Annealing (~60C) dNTPs
4. Extension (~60C)
5. Measure amount of PCR Product Primers (2)
6. Repeat (~95C)

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Introduction To Real-Time Quantitative PCR (qPCR)
 Reporter chemistries

Real-Time qPCR Fluorescence Chemistry

Others, such as hybridization

DNA binding agents Hydrolysis Probes
probes
SYBR® I Dye Dual-labeled Hydrolysis Molecular beacon and
(Taqman®) probe scorpion probes

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Introduction To Real-Time Quantitative PCR (qPCR)
 Reporter chemistries: SYBR® Green I Assay

 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 DNAemits fluorescence very brightly

Fluorescent SYBR I

 The SYBR I signal intensities correlate with DNA amplified (amplicon amount) thus the initialsample input
amounts

Simple & cost saving; high specificity is required since SYBR I binds all
double-strand DNA (non-specific or primer dimer)

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Introduction To Real-Time Quantitative PCR (qPCR)
 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

Each amplicon needs a sequence-specific probe; increased cost & time

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Introduction To Real-Time Quantitative PCR (qPCR)
 Reporter Chemistries: Understanding Kinetics in PCR

Amplification Plot
Plateau phase
(Linear scale) • 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)

Exponential Phase
• Real time PCR does early phase detection
• proportional to input amounts
• 2n=dilution factor

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Introduction To Real-Time Quantitative PCR (qPCR)
 Reporter Chemistries: Understanding Kinetics in PCR

Amplification Plot
Plateau phase
(Linear scale) • End-point PCR data collection at plateau (gel
Plateau analysis)
• Reactions varying due to reagent depletion &
decreased PCR efficiencies (enzyme activity, more product
competing for primer annealing)
Fluorescence
Signal

6 5
107 10 10 Exponential Phase
• Real time PCR does early phase detection
• proportional to input amounts
• 2n=dilution factor

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Introduction To Real-Time Quantitative PCR (qPCR)
 Agenda

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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Introduction To Real-Time Quantitative PCR (qPCR)
 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 eachcycle)

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
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Introduction To Real-Time Quantitative PCR (qPCR)
 Characteristics of a good qPCR Assay: AmplificationEfficiency

Amplification Efficiency: reliable and accurate experiment (Two Methods)

Standard curve
• X axis – dilution
• Y axis - Ct value
• Amp efficiency = 10(-1/slope) -1 *100

Single curve analysis

• PCR Miner:
http://miner.ewindup.info/version2
• “DART”: www.gene-
quantification.de/DART_PCR_version_1.0
.xls

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Introduction To Real-Time Quantitative PCR (qPCR)
 Characteristics of a good qPCR Assay: Sensitivity

Sensitivity
How many copies can my assay detect?
• Important for low expressed genes
or where there is limited sample

Two Methods:
• 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)
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Introduction To Real-Time Quantitative PCR (qPCR)
 Characteristics of a good qPCR Assay: AmplificationEfficiency

Specificity:
1 target amplified

Two Methods:
Melt Curve analysis
• 1 peak, 1 product
Agarose gel
• Band at correct size

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Introduction To Real-Time Quantitative PCR (qPCR)
 Characteristics of a good qPCR Assay: Specificity
Introduction To Real-Time Quantitative PCR
(qPCR)

Melting Curve Analysis

Plot - Normalized
Reporter
Fluorescence

Rn

50%
Normalized

Signal

f luorescence
drop

Gene A Tm: Gene B Tm:

77.36 78.94
Temperatur
e
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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.

40
 Characteristics of a good qPCR Assay: Specificity

Melting Curve Analysis


Plot -1st negative Derivative Single melt curve of each
Reporter
amplicon is required for speci
validation
rate) -delta F/delta T (the
change

Gene B Tm:
78.94
Gene A Tm:
77.36

Temperatur
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e
Introduction To Real-Time Quantitative PCR (qPCR) 41
 Agenda

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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Introduction To Real-Time Quantitative PCR (qPCR)
 Analyzing qPCR curves: How to Define Baseline

Linear Automated Baseline Option

Amplification Plot If an instrument has an adaptive
baseline function

Manual Baseline Option

Use linear view of the plot
C • Set up the baseline reading from cycle #2 to the
Baseli cycle that is 2 cycles before the earliest visible
t amplification
ne
• Usually a baseline falls in 3-15 cycles

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Introduction To Real-Time Quantitative PCR (qPCR)
 Analyzing qPCR curves: How to Define Thershold

Log View Define the Threshold

Amplification Plot 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 ofthe

linear phase of amplification

• Linear phase = exponential phase

• Different runs across samples for the same

experiments should have the same threshold for
comparison

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Introduction To Real-Time Quantitative PCR (qPCR)
 Agenda

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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Introduction To Real-Time Quantitative PCR (qPCR)
 Data Analysis: Biological replicates and technical replicates

Replicates

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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Introduction To Real-Time Quantitative PCR (qPCR)
 Data Analysis: Housekeeping/Reference Genes

Any
changes?

GOI in control GOI in treated

cells cells
Ref Gene in control Ref Gene in drug
cells treated cells
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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Introduction To Real-Time Quantitative PCR (qPCR)
 Data Analysis: Commonly Used Housekeeping Genes

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Introduction To Real-Time Quantitative PCR (qPCR)
 Data & analysis

1. Average Ct values for all gene replicates

2. Calculate Ct value between GOI and HKG for each experiment

3. Average Ct values between experiments (replicates)

4. Calculate Ct values ( Ct experiment- Ct control)

5. Calculate Fold Change 2(-ΔΔCt)

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Introduction To Real-Time Quantitative PCR (qPCR)
 Data & analysis
Treated Cells Un-treated Cells

RNA Isolation RNA Isolation

∆Ct = Ct (GOI -treated) – Ct (HKG -treated)

∆Ct = Ct (GOI -control) – Ct (HKG -control)

∆∆ Ct = ∆Ct (treated) – ∆Ct (control)

Normalized target gene expression level = 2(-∆∆Ct)

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Introduction To Real-Time Quantitative PCR (qPCR)
 Data & analysis: Delta Delta Ct Method - Amplification Plots

GAPDH
Ref
GOI

TNFa

C
Ct tCt

∆∆Ct = ∆Ct (TNFαtreat-GAPDHtreat) - ∆Ct (TNFαcontrol-GAPDHcontrol)

The fold change = 2(-∆∆Ct)

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Introduction To Real-Time Quantitative PCR (qPCR)
 Data & analysis

17.1 17.2 17.2 qPCR replicates

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)

TNFa is up-regulated 32 fold in the treated cells versus the control

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Introduction To Real-Time Quantitative PCR (qPCR)
 Data & analysis

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Introduction To Real-Time Quantitative PCR (qPCR)
 Data Analysis Tools

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Introduction To Real-Time Quantitative PCR (qPCR)
 Want to learn more?

Visit our new Biomarker Insights Blog!

http://biomarkerinsights.qiagen.com/
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• “Long non-coding RNA (lncRNA) as novel biomarkers in colorectal cancer”

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◦ Data Analysis Portal
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Introduction To Real-Time Quantitative PCR (qPCR)
 Agenda

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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Introduction To Real-Time Quantitative PCR (qPCR)
 5
7 Thank you for attending!

Questions?

Contact QIAGEN
[email protected]

Vishwadeepak Tripathi, PhD

[email protected]

More information is also available at:

www.qiagen.com

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Introduction To Real-Time Quantitative PCR (qPCR)