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Introduction To Real-Time Quantitative PCR (qPCR)

SABiosciences, A QIAGEN Company
www.sabiosciences.com

Samuel Rulli, Ph.D.
Samuel.Rulli@QIAGEN.com
support@sabiosciences.com (US)
SAB.EU@qiagen.com (international)
Sample & Assay Technologies
Why qPCR and how does it work?

Car 1
Car 2

Question: How far apart are the 2 cars?
Cars race at same speed to finish line in NYC
As car 1 crosses finish line, calculate time for car 2 to finish
Calculate difference in starting position mathematically (d = rate x time)

-2-

Sample & Assay Technologies
Why qPCR and how does it work?

Car 1
Car 2

Question: How far apart are the 2 cars?
Cars race at same speed to finish line in NYC
As car 1 crosses finish line, calculate time for car 2 to finish
Calculate difference in starting position mathematically (d = rate x time)

-3-

Sample & Assay Technologies
Why qPCR and how does it work?

Threshold line
Sample 1
Sample 2

Question: What is the difference in the amount of the same gene in 2 samples?
Amplify gene at same to finish line (threshold)
As sample 1 crosses threshold line, calculate time (cycles) for sample 2 to finish
Calculate difference in starting amounts mathematically (∆Ct)

-4-

Sample & Assay Technologies
The Seminar Topics
What is qPCR? Applications and workflow
qPCR for gene expression: What is the change in gene
expression during differentiation?
Factors influencing the performance of a qPCR assay
RNA purity and integrity
Reverse Transcription
qPCR, reporter chemistries and characteristics of a good qPCR
assay
Analyzing qPCR curves
Data & analysis
-5-

Sample & Assay Technologies
What does Real-Time qPCR Stands for?

Real-time qPCR is a sensitive and reliable method for
detection and quantification of nucleic acids (DNA,& RNA
(cDNA) levels.
It is based on detection and quantification of fluorescence
emitted from a reporter molecule at real time.
This detection occurs during the accumulation of the PCR
product with each cycle of amplification, thus allows
monitoring 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.
-6-

Sample & Assay Technologies
Applications for qPCR

RNA

Gene Expression Profiling Analysis
miRNA Expression Profiling Analysis
SNP Genotyping & allelic discrimination
Somatic Mutation Analysis

DNA

Copy Number Detection/Variation Analysis
Chromatin IP Quantification
DNA Methylation Detection
Pathogen Detection
Viral Quantification

-7-

Sample & Assay Technologies
Work Flow: A Brief Look
Samples

SYBR® or Probe
RNA (total, mRNA, small RNA)

DNA

Reverse transcription

cDNA

Assay Design
Sample quality
control
Assay Optimization
Real Time PCR Set Up
Instrument Set up & thermal cycling

Data Output & Analysis

*
-8-

Sample & Assay Technologies
Applications for qPCR: gene expression profiling

RNA

Gene Expression Profiling Analysis
miRNA Expression Profiling Analysis
SNP Genotyping & allelic discrimination
Somatic Mutation Analysis

DNA

Copy Number Detection/Variation Analysis
Chromatin IP Quantification
DNA Methylation Detection
Pathogen Detection
Viral Quantification

-9-

Sample & Assay Technologies
Application example:
gene expression changes during differentiation
Osteogenesis – Day 16
T4
T3

T2
hMSC

T1

Neurogenesis – 72 hr
T1

T2
T3

T4

Differentiation protocol
Collect Total RNA at different time points
Measure 1 HKG and 1 GOI (TNFα)
Repeat experiment 3x (biological replicates)
- 10 -

Sample & Assay Technologies
Work Flow: Gene expression profiling
Samples

SYBR®
RNA (total, RNA)
Reverse transcription

cDNA

Assay Design
Sample quality
control
Assay Optimization
Real Time PCR Set Up
Instrument Set up & thermal cycling

Data Output & Analysis
- 11 -

Sample & Assay Technologies
Factors Critical For A Successful Assay
DNA or RNA sample preparation --- Template quality
- Choose appropriate sample preparation kits/reagents (inhibitors can
compromise RT or PCR Reaction
Reverse transcription for converting RNA to cDNA
- Choose RT kits (type of RT, which type of primers, controls?)
Assay design: chemistry, specificity, PCR efficiency, & throughput & cost
- Choose validated assay, or need to validate our own?
Running PCR
- Choose commercial mastermix or make own (primer, probe, master mix)
Data analysis tool
- User friendly & streamlined data analysis module

- 12 -

Sample & Assay Technologies
RNA Isolation
RNA Isolation:
Qiazol?
Column based method (RNeasy?)
Both: Efficient lysis and inhibition of RNases; molecular grade RNA
miRNA? Use a kit specific for miRNA and mRNA
Qiazol:
phenol/guanidine-based lysis
Instant inactivation of RNases
Instant end of biological activities
Column cleanup:
Molecular biology grade RNA

RNeasy Lipid tissue mini Kit
- 13 -

Sample & Assay Technologies
RNA Sample Quality
Spectroscopic: measure 260/280 and 230/280
OD260 is used to calculate amount of nucleic acid
260/280 ratio (typical minimum value 1.8)
260/230 ratio (typical minimum value 1.7)
Low ratio may indicate protein, QIAzol, Carbohydrates, Guanidine HCL,
Absorbance measurements do not show integrity of RNA
Denaturing RNA Agarose Gel
Used to detect integrity of RNA (usually through ribosomal bands)
QIAxcel
Automate
RNA integrity
analysis

- 14 -

Sample & Assay Technologies
qPCR Components & Steps: Overview
B. Primers/Probes

A. Templates
10 – 1000 copies of nucleic acids
100 pg to 1 µg RNA

C. Master Mix
DNA Polymerase
Mg++
dNTP
Buffer
*Passive reference dye

Reverse Transcription

Two-step qPCR: (1) RT (2) qPCR
One-step qPCR: one tube reaction

Denaturation
Denaturation

Annealing

Extension

Annealing/Extension
- 15 -

Sample & Assay Technologies
qPCR Components & Steps: Overview
B. Primers/Probes

A. Templates
10 – 1000 copies of nucleic acids
100 pg to 1 µg RNA

C. Master Mix
DNA Polymerase
Mg++
dNTP
Buffer
*Passive reference dye

Reverse Transcription

Two-step qPCR: (1) RT (2) qPCR
One-step qPCR: one tube reaction

Denaturation
Denaturation

Annealing

Extension

Annealing/Extension
- 16 -

Sample & Assay Technologies
Reverse Transcription

Used to make cDNA copy of RNA
Reagents:
Reverse transcriptase – many different kinds
dNTPs
Buffers for RT
Primers
Random pentamers or hexamers?
Oligo-dT?
Both?
Control RNA to monitor reverse transcription kit?
Note:

Make sure that RT reaction is linear
Do not try to reverse transcript too much RNA
Sensitivity of qPCR step is dependent on good RT reaction
Monitor RT reaction to ensure equal RT efficiency across all samples

- 17 -

Sample & Assay Technologies
What is in a PCR Reaction?

PCR= Polymerase Chain Reaction
Exponential Amplification of DNA in single tube

DNA Template
(ss or ds)

Polymerase

“thermostable” i.e. can withstand temperatures
Up to ~95C
All reagents in
Excess (non-limiting)

dNTPs.
Primers (2)

- 18 -

Sample & Assay Technologies
PCR Reaction in Action

DNA Template
(ss or ds)

Polymerase

dNTPs.
Primers (2)

1.
2.
3.
4.
- 19 -

Heat denature template (~95C)
Anneal Primer (~60C)
Extend primer (~60C)
Repeat (~95C)
Sample & Assay Technologies
PCR Reaction in Action

Heat denature
DNA Template
(ss or ds)

Polymerase

dNTPs.
Primers (2)

1.
2.
3.
4.
- 20 -

Heat denature template (~95C)
Anneal Primer (~60C)
Extend primer (~60C)
Repeat (~95C)
Sample & Assay Technologies
PCR Reaction in Action

DNA Template
(ss or ds)

Polymerase

dNTPs.

1.
2.
3.
4.
- 21 -

Heat denature template (~95C)
Anneal Primer (~60C)
Extend primer (~60C)
Repeat (~95C)
Sample & Assay Technologies
PCR Reaction in Action

Polymerase

Polymerase

DNA Template
(ss or ds)

Polymerase

dNTPs.

1.
2.
3.
4.
- 22 -

Heat denature template (~95C)
Anneal Primer (~60C)
Extend primer (~60C)
Repeat (~95C)
Sample & Assay Technologies
PCR Reaction in Action

Polymerase

DNA Template
(ss or ds)

Polymerase

Polymerase

dNTPs.

1.
2.
3.
4.
- 23 -

Heat denature template (~95C)
Anneal Primer (~60C)
Extend primer (~60C)
Repeat (~95C)
Sample & Assay Technologies
PCR Reaction in Action

Polymerase

DNA Template
(ss or ds)
Polymerase
Polymerase

dNTPs.

1.
2.
3.
4.
- 24 -

Heat denature template (~95C)
Anneal Primer (~60C)
Extend primer (~60C)
Repeat (~95C)
Sample & Assay Technologies
PCR Reaction in Action

Polymerase

DNA Template
(ss or ds)

dNTPs.

1.
2.
3.
4.
- 25 -

Heat denature template (~95C)
Anneal Primer (~60C)
Extend primer (~50 to ~70C)
Repeat (~95C)
Sample & Assay Technologies
qPCR Reaction: Measure DNA amount at end of each cycle to
get ratio of DNA or absolute amount (if using a standard)

Polymerase

DNA Template
(ss or ds)

dNTPs.

1.
2.
3.
4.
5.

Heat denature template (~95C)
Anneal Primer (~60C)
Extend primer (~50 to ~70C)
Measure Amount of PCR Product
Repeat (~95C)
- 26 -

Sample & Assay Technologies
Real-Time qPCR Fluorescence Chemistry

DNA binding agents
Two most commonly used
chemistries in qPCR community

SYBR® I Dye,
Hydrolysis Probes

Dual-labeled Hydrolysis (Taqman®) probe
Others, such as hybridization probes
-Molecular beacon and scorpion probes

- 27 -

Sample & Assay Technologies
SYBR® Green I Assay: Fluorescent DNA Binding Dye
Non 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
Simple & cost saving
Fluorescent SYBR I

The SYBR I signal intensities correlate with
DNA amplified (amplicon amount) thus the
initial sample input amounts
High Specificity Is Required when using SYBR Green
since SYBR I binds all double-strand DNA (non-specific or primer dimmer).
- 28 -

Sample & Assay Technologies
Understanding Kinetics in PCR

Amplification Plot (Linear scale)

Fluorescence Signal

Plateau

End point data collection
at plateau (gel analysis)

Reactions start varying due to
reagent depletion & decreased
PCR efficiencies (enzyme activity,
More product competing for primer
annealing

107 106 105

Real time PCR does early phase detection at
the exponential state

Precisely proportional to input amounts
- 29 -

Sample & Assay Technologies
Hydrolysis Based Probe - - - Taqman® 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 (cost & time)
- 30 -

Sample & Assay Technologies
Understanding Kinetics in PCR

Amplification Plot (Linear scale)

Fluorescence Signal

Plateau

End point data collection
at plateau (gel analysis)

Reactions start varying due to
reagent depletion & decreased
PCR efficiencies (enzyme activity,
More product competing for primer
annealing

107 106 105

Real time PCR does early phase detection at
the exponential state

Precisely proportional to input amounts
- 31 -

Sample & Assay Technologies
Characteristics of a good qPCR Assay

Amplification efficiency: 100% during exponential phase
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)

- 32 -

Sample & Assay Technologies
Amplification Analysis: standard curve and single curve
analysis
Plot:
x axis dilution
Y axis Ct value
Amp efficiency = 10(-1/slope) -1 *100

Single curve analysis
PCR Miner
http://miner.ewindup.info/versio
n2
“DART”
www.genequantification.de/DART_PCR_
version_1.0.xls
- 33 -

Sample & Assay Technologies
Sensitivity: How many copies can my assay detect?
Sensitivity is very 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)

- 34 -

Sample & Assay Technologies
Specificity: SYBR Green

Single peak dissociation
curves
Single gel bands of
predicted size

- 35 -

Sample & Assay Technologies
Melt Curve Analysis: The General Program Steps

Rapid heating of amplified samples to 94°C to denature the
DNA
Cooling the sample to 60°C to let DNA double strands anneal
Slowly heating (by increasing the temperature, usually
0.2°C/sec) the sample while plotting the fluorescent signal
versus temperature.
As the temperature increases, and DNA melts, the fluorescent
signal should decrease.
There will be a significant drop of the signal when 50% DNA
melts.

- 36 -

Sample & Assay Technologies
Melting Curve Analysis --- Normalized Reporter Plot

Samples

Tm

Gene A

77.36

Gene B

Rn

Normalized Fluorescence Signal

Plot - Normalized Reporter (Fluorescence/Passive dye signal)

78.94

50% fluorescence
drop

Tm: B

Tm: A
Temperature
- 37 -

Sample & Assay Technologies
Melt Curve Analysis --- 1st Negative Derivative Plot

-delta F/delta T (the change rate)

Plot - - -1st negative Derivative Reporter
Gene B

Gene A

Single melt curve of each amplicon
is required for specificity validation!

Tm: B
Tm: A

Temperature
- 38 -

Sample & Assay Technologies
Biological replicates are better than technical replicates

Biological Replicates: 3 different experiments
Shows variability due to experiment

Technical replicates: 3 different measurements for same step
Shows variability due to pipetting, machine, enzymes, etc.

Sacrifice Technical replicates for biological replicates, always do at least 3 to
get fold change and p value (or other statistics such as 95% confidence interval)

- 39 -

Sample & Assay Technologies
Thermal Cycling Programs

1

Instrument default melt curve program

Step 1
Activation
Step 2
Data
collection

Melt curve analysis
(SYBR Only)

Stratagene Mxp3005p
Step 3
Melt curve
analysis

- 40 -

Sample & Assay Technologies
Run qPCR - - - Results

- 41 -

Sample & Assay Technologies
How To Define/Set Up The Baseline

Linear Amplification Plot
-Automated Baseline Option
if an instrument has a adaptive baseline
function

-Define manually:
(1) Use linear view of the plot

Baseline

Ct

(2) Set up the baseline reading from
cycle #2 to the cycle that 2 cycles before
the earliest visible amplification
(3) Usually a baseline falls in 3-15 cycles

- 42 -

Sample & Assay Technologies
How To Define Threshold

Log View 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
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

- 43 -

Sample & Assay Technologies
Reference Genes (Housekeeping Genes)
For Normalization
Any changes?
Gene of interest A in untreated cells

GOI A in drug treated cells

Reference Gene B in untreated cells

Ref Gene B in drug treated cells

The expression level of a reference gene remain consistent under
experimental conditions or different tissues

A Reference Gene is 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

- 44 -

Sample & Assay Technologies
Commonly Used Housekeeping Genes

- 45 -

Sample & Assay Technologies
Data Analysis website

1.) Average Ct values for all gene replicates
2.) Calculate Delta Ct value between GOI and HKG for each experiment
3.) Average Delta Ct values between experiments (replicates)
4.) Calculate Delta-Delta Ct values ( Delta Ct experiment- Delta Ct control)
5.) Calculate Fold Change 2(-Delta Delta Ct)

- 46 -

Sample & Assay Technologies
Normalized Gene Expression Level
Any changes?
Target Gene A in control cells

Target Gene A in drug treated cells

Reference Gene B in control cells

Ref Gene B in drug treated cells

∆Ct = Ct (Target A -treated) – Ct (Ref B-treated)
∆Ct = Ct (Target A-control) – Ct (Ref B-control)
∆∆ Ct = ∆ Ct (treated) – Ct (control)

Normalized target gene expression level =

- 47 -

2(-∆∆Ct)
Sample & Assay Technologies
Delta Delta Ct Method: A Look of Amplification Plots

GAPDH

Ref
GOI

TNFα
α

Ct

Ct

Ct

Ct

∆∆Ct = ∆Ct (TNFαtreat-GAPDHtreat) - ∆ct (TNFαcontrol-GAPDHcontrol)
The fold change = 2(-∆∆Ct)
- 48 -

Sample & Assay Technologies
Data Analysis website
1.) Average Ct values for all gene replicates

2.) Calculate Delta Ct value: GOI-HKG

3.) Average Delta Ct values between experiments (replicates)

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

5.) Calculate Fold Change 2(-Delta Delta Ct)

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

- 49 -

Sample & Assay Technologies
Data Analysis website
qPCR replicates

17.1, 17.2, 17.2
1.) Average Ct values for all gene replicates

2.) Calculate Delta Ct value: GOI-HKG

3.) Average Delta Ct values between experiments (replicates)

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

5.) Calculate Fold Change 2(-Delta Delta Ct)

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

- 50 -

Sample & Assay Technologies
http://www.sabiosciences.com/dataanalysis.php

- 51 -

Sample & Assay Technologies
http://www.sabiosciences.com/dataanalysis.php

- 52 -

Sample & Assay Technologies
Questions?

Ask now or contact Technical Support M – F, 9 AM – 6 PM EST
Telephone: (888) 503-3187
Email: support@SABiosciences.com
SAB.EU@qiagen.com
Thank you!

Sample & Assay Technologies

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Q pcr introduction 2013

  • 1. Introduction To Real-Time Quantitative PCR (qPCR) SABiosciences, A QIAGEN Company www.sabiosciences.com Samuel Rulli, Ph.D. Samuel.Rulli@QIAGEN.com support@sabiosciences.com (US) SAB.EU@qiagen.com (international) Sample & Assay Technologies
  • 2. Why qPCR and how does it work? Car 1 Car 2 Question: How far apart are the 2 cars? Cars race at same speed to finish line in NYC As car 1 crosses finish line, calculate time for car 2 to finish Calculate difference in starting position mathematically (d = rate x time) -2- Sample & Assay Technologies
  • 3. Why qPCR and how does it work? Car 1 Car 2 Question: How far apart are the 2 cars? Cars race at same speed to finish line in NYC As car 1 crosses finish line, calculate time for car 2 to finish Calculate difference in starting position mathematically (d = rate x time) -3- Sample & Assay Technologies
  • 4. Why qPCR and how does it work? Threshold line Sample 1 Sample 2 Question: What is the difference in the amount of the same gene in 2 samples? Amplify gene at same to finish line (threshold) As sample 1 crosses threshold line, calculate time (cycles) for sample 2 to finish Calculate difference in starting amounts mathematically (∆Ct) -4- Sample & Assay Technologies
  • 5. The Seminar Topics What is qPCR? Applications and workflow qPCR for gene expression: What is the change in gene expression during differentiation? Factors influencing the performance of a qPCR assay RNA purity and integrity Reverse Transcription qPCR, reporter chemistries and characteristics of a good qPCR assay Analyzing qPCR curves Data & analysis -5- Sample & Assay Technologies
  • 6. What does Real-Time qPCR Stands for? Real-time qPCR is a sensitive and reliable method for detection and quantification of nucleic acids (DNA,& RNA (cDNA) levels. It is based on detection and quantification of fluorescence emitted from a reporter molecule at real time. This detection occurs during the accumulation of the PCR product with each cycle of amplification, thus allows monitoring 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. -6- Sample & Assay Technologies
  • 7. Applications for qPCR RNA Gene Expression Profiling Analysis miRNA Expression Profiling Analysis SNP Genotyping & allelic discrimination Somatic Mutation Analysis DNA Copy Number Detection/Variation Analysis Chromatin IP Quantification DNA Methylation Detection Pathogen Detection Viral Quantification -7- Sample & Assay Technologies
  • 8. Work Flow: A Brief Look Samples SYBR® or Probe RNA (total, mRNA, small RNA) DNA Reverse transcription cDNA Assay Design Sample quality control Assay Optimization Real Time PCR Set Up Instrument Set up & thermal cycling Data Output & Analysis * -8- Sample & Assay Technologies
  • 9. Applications for qPCR: gene expression profiling RNA Gene Expression Profiling Analysis miRNA Expression Profiling Analysis SNP Genotyping & allelic discrimination Somatic Mutation Analysis DNA Copy Number Detection/Variation Analysis Chromatin IP Quantification DNA Methylation Detection Pathogen Detection Viral Quantification -9- Sample & Assay Technologies
  • 10. Application example: gene expression changes during differentiation Osteogenesis – Day 16 T4 T3 T2 hMSC T1 Neurogenesis – 72 hr T1 T2 T3 T4 Differentiation protocol Collect Total RNA at different time points Measure 1 HKG and 1 GOI (TNFα) Repeat experiment 3x (biological replicates) - 10 - Sample & Assay Technologies
  • 11. Work Flow: Gene expression profiling Samples SYBR® RNA (total, RNA) Reverse transcription cDNA Assay Design Sample quality control Assay Optimization Real Time PCR Set Up Instrument Set up & thermal cycling Data Output & Analysis - 11 - Sample & Assay Technologies
  • 12. Factors Critical For A Successful Assay DNA or RNA sample preparation --- Template quality - Choose appropriate sample preparation kits/reagents (inhibitors can compromise RT or PCR Reaction Reverse transcription for converting RNA to cDNA - Choose RT kits (type of RT, which type of primers, controls?) Assay design: chemistry, specificity, PCR efficiency, & throughput & cost - Choose validated assay, or need to validate our own? Running PCR - Choose commercial mastermix or make own (primer, probe, master mix) Data analysis tool - User friendly & streamlined data analysis module - 12 - Sample & Assay Technologies
  • 13. RNA Isolation RNA Isolation: Qiazol? Column based method (RNeasy?) Both: Efficient lysis and inhibition of RNases; molecular grade RNA miRNA? Use a kit specific for miRNA and mRNA Qiazol: phenol/guanidine-based lysis Instant inactivation of RNases Instant end of biological activities Column cleanup: Molecular biology grade RNA RNeasy Lipid tissue mini Kit - 13 - Sample & Assay Technologies
  • 14. RNA Sample Quality Spectroscopic: measure 260/280 and 230/280 OD260 is used to calculate amount of nucleic acid 260/280 ratio (typical minimum value 1.8) 260/230 ratio (typical minimum value 1.7) Low ratio may indicate protein, QIAzol, Carbohydrates, Guanidine HCL, Absorbance measurements do not show integrity of RNA Denaturing RNA Agarose Gel Used to detect integrity of RNA (usually through ribosomal bands) QIAxcel Automate RNA integrity analysis - 14 - Sample & Assay Technologies
  • 15. qPCR Components & Steps: Overview B. Primers/Probes A. Templates 10 – 1000 copies of nucleic acids 100 pg to 1 µg RNA C. Master Mix DNA Polymerase Mg++ dNTP Buffer *Passive reference dye Reverse Transcription Two-step qPCR: (1) RT (2) qPCR One-step qPCR: one tube reaction Denaturation Denaturation Annealing Extension Annealing/Extension - 15 - Sample & Assay Technologies
  • 16. qPCR Components & Steps: Overview B. Primers/Probes A. Templates 10 – 1000 copies of nucleic acids 100 pg to 1 µg RNA C. Master Mix DNA Polymerase Mg++ dNTP Buffer *Passive reference dye Reverse Transcription Two-step qPCR: (1) RT (2) qPCR One-step qPCR: one tube reaction Denaturation Denaturation Annealing Extension Annealing/Extension - 16 - Sample & Assay Technologies
  • 17. Reverse Transcription Used to make cDNA copy of RNA Reagents: Reverse transcriptase – many different kinds dNTPs Buffers for RT Primers Random pentamers or hexamers? Oligo-dT? Both? Control RNA to monitor reverse transcription kit? Note: Make sure that RT reaction is linear Do not try to reverse transcript too much RNA Sensitivity of qPCR step is dependent on good RT reaction Monitor RT reaction to ensure equal RT efficiency across all samples - 17 - Sample & Assay Technologies
  • 18. What is in a PCR Reaction? PCR= Polymerase Chain Reaction Exponential Amplification of DNA in single tube DNA Template (ss or ds) Polymerase “thermostable” i.e. can withstand temperatures Up to ~95C All reagents in Excess (non-limiting) dNTPs. Primers (2) - 18 - Sample & Assay Technologies
  • 19. PCR Reaction in Action DNA Template (ss or ds) Polymerase dNTPs. Primers (2) 1. 2. 3. 4. - 19 - Heat denature template (~95C) Anneal Primer (~60C) Extend primer (~60C) Repeat (~95C) Sample & Assay Technologies
  • 20. PCR Reaction in Action Heat denature DNA Template (ss or ds) Polymerase dNTPs. Primers (2) 1. 2. 3. 4. - 20 - Heat denature template (~95C) Anneal Primer (~60C) Extend primer (~60C) Repeat (~95C) Sample & Assay Technologies
  • 21. PCR Reaction in Action DNA Template (ss or ds) Polymerase dNTPs. 1. 2. 3. 4. - 21 - Heat denature template (~95C) Anneal Primer (~60C) Extend primer (~60C) Repeat (~95C) Sample & Assay Technologies
  • 22. PCR Reaction in Action Polymerase Polymerase DNA Template (ss or ds) Polymerase dNTPs. 1. 2. 3. 4. - 22 - Heat denature template (~95C) Anneal Primer (~60C) Extend primer (~60C) Repeat (~95C) Sample & Assay Technologies
  • 23. PCR Reaction in Action Polymerase DNA Template (ss or ds) Polymerase Polymerase dNTPs. 1. 2. 3. 4. - 23 - Heat denature template (~95C) Anneal Primer (~60C) Extend primer (~60C) Repeat (~95C) Sample & Assay Technologies
  • 24. PCR Reaction in Action Polymerase DNA Template (ss or ds) Polymerase Polymerase dNTPs. 1. 2. 3. 4. - 24 - Heat denature template (~95C) Anneal Primer (~60C) Extend primer (~60C) Repeat (~95C) Sample & Assay Technologies
  • 25. PCR Reaction in Action Polymerase DNA Template (ss or ds) dNTPs. 1. 2. 3. 4. - 25 - Heat denature template (~95C) Anneal Primer (~60C) Extend primer (~50 to ~70C) Repeat (~95C) Sample & Assay Technologies
  • 26. qPCR Reaction: Measure DNA amount at end of each cycle to get ratio of DNA or absolute amount (if using a standard) Polymerase DNA Template (ss or ds) dNTPs. 1. 2. 3. 4. 5. Heat denature template (~95C) Anneal Primer (~60C) Extend primer (~50 to ~70C) Measure Amount of PCR Product Repeat (~95C) - 26 - Sample & Assay Technologies
  • 27. Real-Time qPCR Fluorescence Chemistry DNA binding agents Two most commonly used chemistries in qPCR community SYBR® I Dye, Hydrolysis Probes Dual-labeled Hydrolysis (Taqman®) probe Others, such as hybridization probes -Molecular beacon and scorpion probes - 27 - Sample & Assay Technologies
  • 28. SYBR® Green I Assay: Fluorescent DNA Binding Dye Non 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 Simple & cost saving Fluorescent SYBR I The SYBR I signal intensities correlate with DNA amplified (amplicon amount) thus the initial sample input amounts High Specificity Is Required when using SYBR Green since SYBR I binds all double-strand DNA (non-specific or primer dimmer). - 28 - Sample & Assay Technologies
  • 29. Understanding Kinetics in PCR Amplification Plot (Linear scale) Fluorescence Signal Plateau End point data collection at plateau (gel analysis) Reactions start varying due to reagent depletion & decreased PCR efficiencies (enzyme activity, More product competing for primer annealing 107 106 105 Real time PCR does early phase detection at the exponential state Precisely proportional to input amounts - 29 - Sample & Assay Technologies
  • 30. Hydrolysis Based Probe - - - Taqman® 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 (cost & time) - 30 - Sample & Assay Technologies
  • 31. Understanding Kinetics in PCR Amplification Plot (Linear scale) Fluorescence Signal Plateau End point data collection at plateau (gel analysis) Reactions start varying due to reagent depletion & decreased PCR efficiencies (enzyme activity, More product competing for primer annealing 107 106 105 Real time PCR does early phase detection at the exponential state Precisely proportional to input amounts - 31 - Sample & Assay Technologies
  • 32. Characteristics of a good qPCR Assay Amplification efficiency: 100% during exponential phase 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) - 32 - Sample & Assay Technologies
  • 33. Amplification Analysis: standard curve and single curve analysis Plot: x axis dilution Y axis Ct value Amp efficiency = 10(-1/slope) -1 *100 Single curve analysis PCR Miner http://miner.ewindup.info/versio n2 “DART” www.genequantification.de/DART_PCR_ version_1.0.xls - 33 - Sample & Assay Technologies
  • 34. Sensitivity: How many copies can my assay detect? Sensitivity is very 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) - 34 - Sample & Assay Technologies
  • 35. Specificity: SYBR Green Single peak dissociation curves Single gel bands of predicted size - 35 - Sample & Assay Technologies
  • 36. Melt Curve Analysis: The General Program Steps Rapid heating of amplified samples to 94°C to denature the DNA Cooling the sample to 60°C to let DNA double strands anneal Slowly heating (by increasing the temperature, usually 0.2°C/sec) the sample while plotting the fluorescent signal versus temperature. As the temperature increases, and DNA melts, the fluorescent signal should decrease. There will be a significant drop of the signal when 50% DNA melts. - 36 - Sample & Assay Technologies
  • 37. Melting Curve Analysis --- Normalized Reporter Plot Samples Tm Gene A 77.36 Gene B Rn Normalized Fluorescence Signal Plot - Normalized Reporter (Fluorescence/Passive dye signal) 78.94 50% fluorescence drop Tm: B Tm: A Temperature - 37 - Sample & Assay Technologies
  • 38. Melt Curve Analysis --- 1st Negative Derivative Plot -delta F/delta T (the change rate) Plot - - -1st negative Derivative Reporter Gene B Gene A Single melt curve of each amplicon is required for specificity validation! Tm: B Tm: A Temperature - 38 - Sample & Assay Technologies
  • 39. Biological replicates are better than technical replicates Biological Replicates: 3 different experiments Shows variability due to experiment Technical replicates: 3 different measurements for same step Shows variability due to pipetting, machine, enzymes, etc. Sacrifice Technical replicates for biological replicates, always do at least 3 to get fold change and p value (or other statistics such as 95% confidence interval) - 39 - Sample & Assay Technologies
  • 40. Thermal Cycling Programs 1 Instrument default melt curve program Step 1 Activation Step 2 Data collection Melt curve analysis (SYBR Only) Stratagene Mxp3005p Step 3 Melt curve analysis - 40 - Sample & Assay Technologies
  • 41. Run qPCR - - - Results - 41 - Sample & Assay Technologies
  • 42. How To Define/Set Up The Baseline Linear Amplification Plot -Automated Baseline Option if an instrument has a adaptive baseline function -Define manually: (1) Use linear view of the plot Baseline Ct (2) Set up the baseline reading from cycle #2 to the cycle that 2 cycles before the earliest visible amplification (3) Usually a baseline falls in 3-15 cycles - 42 - Sample & Assay Technologies
  • 43. How To Define Threshold Log View 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 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 - 43 - Sample & Assay Technologies
  • 44. Reference Genes (Housekeeping Genes) For Normalization Any changes? Gene of interest A in untreated cells GOI A in drug treated cells Reference Gene B in untreated cells Ref Gene B in drug treated cells The expression level of a reference gene remain consistent under experimental conditions or different tissues A Reference Gene is 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 - 44 - Sample & Assay Technologies
  • 45. Commonly Used Housekeeping Genes - 45 - Sample & Assay Technologies
  • 46. Data Analysis website 1.) Average Ct values for all gene replicates 2.) Calculate Delta Ct value between GOI and HKG for each experiment 3.) Average Delta Ct values between experiments (replicates) 4.) Calculate Delta-Delta Ct values ( Delta Ct experiment- Delta Ct control) 5.) Calculate Fold Change 2(-Delta Delta Ct) - 46 - Sample & Assay Technologies
  • 47. Normalized Gene Expression Level Any changes? Target Gene A in control cells Target Gene A in drug treated cells Reference Gene B in control cells Ref Gene B in drug treated cells ∆Ct = Ct (Target A -treated) – Ct (Ref B-treated) ∆Ct = Ct (Target A-control) – Ct (Ref B-control) ∆∆ Ct = ∆ Ct (treated) – Ct (control) Normalized target gene expression level = - 47 - 2(-∆∆Ct) Sample & Assay Technologies
  • 48. Delta Delta Ct Method: A Look of Amplification Plots GAPDH Ref GOI TNFα α Ct Ct Ct Ct ∆∆Ct = ∆Ct (TNFαtreat-GAPDHtreat) - ∆ct (TNFαcontrol-GAPDHcontrol) The fold change = 2(-∆∆Ct) - 48 - Sample & Assay Technologies
  • 49. Data Analysis website 1.) Average Ct values for all gene replicates 2.) Calculate Delta Ct value: GOI-HKG 3.) Average Delta Ct values between experiments (replicates) 4.) Calculate Delta-Delta Ct values (Delta Ct experiment- Delta Ct control) 5.) Calculate Fold Change 2(-Delta Delta Ct) TNFα is up-regulated 32 fold in the treated cells versus the control - 49 - Sample & Assay Technologies
  • 50. Data Analysis website qPCR replicates 17.1, 17.2, 17.2 1.) Average Ct values for all gene replicates 2.) Calculate Delta Ct value: GOI-HKG 3.) Average Delta Ct values between experiments (replicates) 4.) Calculate Delta-Delta Ct values (Delta Ct experiment- Delta Ct control) 5.) Calculate Fold Change 2(-Delta Delta Ct) TNFα is up-regulated 32 fold in the treated cells versus the control - 50 - Sample & Assay Technologies
  • 53. Questions? Ask now or contact Technical Support M – F, 9 AM – 6 PM EST Telephone: (888) 503-3187 Email: support@SABiosciences.com SAB.EU@qiagen.com Thank you! Sample & Assay Technologies