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Finish
Line
Car 2
Car 1
Finish
Line
Car 2
Car 1
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Seminar Topics
1.
2.
qPCR for gene expression: What is the change in gene expression during
differentiation?
3.
4.
5.
Reverse Transcription
6.
qPCR in Action
7.
Reporter chemistries
8.
9.
Quantitative Polymerase Chain Reaction (qPCR) is a sensitive and reliable method for
detection and quantification of nucleic acid (DNA & RNA) levels.
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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DNA
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Chromatin IP Quantification
Pathogen Detection
Viral Quantification
Sample
RNA (total, mRNA,
small RNA)
DNA
Sample QC
cDNA
SYBR/Probe
Assay design
Assay
Optimization
Sample QC
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DNA
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Chromatin IP Quantification
Pathogen Detection
Viral Quantification
T2
T3
T1
Neurogenesis 72 hr
T1
T2
T3
T4
Sample QC
cDNA
SYBR
Assay
design
Assay
Optimization
Thermocycling
Data Analysis
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Choose RT kits
type of RT
which type of primers
controls?
C. Assay design: chemistry, specificity, PCR efficiency, & throughput & cost
D. Running PCR
User friendly
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RNA Isolation:
Considerations
Sample type/amount
Target (total RNA, mRNA, miRNA)
Throughput
Difficult samples (stool, FFPE)
Challenges
Contamination (gDNA)
Yield
Quality
Method
Qiazol?
Column based method (RNeasy?)
Both: Efficient lysis and inhibition of RNases; molecular grade RNA
miRNA? Use a kit specific for miRNA and mRNA
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Integrity:
QIAxcel/ Bioanalyzer
Capillary electrophoresis
Automate RNA integrity analysis
RNA integrity analysis number
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16
One-Step
PCR
1 Tube Reaction
Two-Step
PCR
2 separate reactions
RT Reaction
qPCR Reaction
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B. Primers/Probes
C. Master Mix
DNA polymerase
Mg++
dNTPs
Buffer
Passive reference dye
D. Cycling Conditions
Denature>Annealing>Extension
Denature>Annealing/Extension
Reverse Transcription
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?
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 efficiency across samples
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qPCR in Action
DNA Template
(ss or ds)
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Polymerase
dNTPs
Primers (2)
qPCR in Action
DNA Template
(ss or ds)
Polymerase
1.
2.
3.
4.
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dNTPs
Primers (2)
qPCR in Action
Heat denature
DNA Template
(ss or ds)
Polymerase
1.
2.
3.
4.
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dNTPs
Primers (2)
qPCR in Action
DNA Template
Polymerase
1.
2.
3.
4.
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dNTPs
Primers (2)
qPCR in Action
Polymerase
Polymerase
DNA Template
(ss or ds)
Polymerase
1.
2.
3.
4.
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dNTPs
Primers (2)
qPCR in Action
Polymerase
DNA Template
(ss or ds)
Polymerase
Polymerase
1.
2.
3.
4.
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dNTPs
Primers (2)
qPCR in Action
Polymerase
DNA Template
(ss or ds)
Polymerase
Polymerase
1.
2.
3.
4.
Sample to Insight
dNTPs
Primers (2)
qPCR in Action
DNA Template
(ss or ds)
Polymerase
1.
2.
3.
4.
Sample to Insight
dNTPs
Primers (2)
qPCR in Action
DNA Template
(ss or ds)
1.
2.
3.
4.
5.
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Polymerase
dNTPs
Primers (2)
Reporter chemistries
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Plateau phase
End-point PCR data
collection at plateau
(gel analysis)
Plateau
Fluorescence Signal
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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
Reporter Chemistries
Hydrolysis Based Probe - - - Taqman Probe Assay
Plateau phase
End-point PCR data
collection at plateau
(gel analysis)
Plateau
Fluorescence Signal
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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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Rn
Temperature
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temperature.
Temperature
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Baseline
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Ct
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Any changes?
GOI A in control 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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GAPDH
Ref
GOI
TNF
Ct
Ct
Ct
Ct
Ct = Ct (TNFtreat-GAPDHtreat) - ct (TNFcontrol-GAPDHcontrol)
The fold change = 2(-Ct)
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qPCR replicates
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2.
qPCR for gene expression: What is the change in gene expression during
differentiation?
3.
4.
5.
Reverse Transcription
6.
qPCR in Action
7.
Reporter chemistries
8.
9.
Register:
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