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Real-Time PCR
mRNA quantification
What do mRNA levels tell us?
DNAmRNAprotein
• Reflect level of gene expression
• Information about cell response
• Protein production (not always)
quantitative mRNA/DNA analysis
Direct
-Northern blotting
-In situ hybridization
PCR amplification
-Regular RT-PCR
-Real time PCR
(Microarrays)
Nomenclature
RT-PCR = Reverse Transcriptase PCR
qReal time PCR = quantitative Real-Time PCR
RT-PCR
• Isolate RNA
• cDNA synthesis
• PCR reaction
Why isn´t this good enough?
What’s Wrong With
Agarose Gels?
*
*
*
*
*
•
•
Low sensitivity
Low resolution
Non-automated
Size-based discrimination only
Results are not expressed as numbers
 based on personal evaluation
Ethidium bromide staining is not very
quantitative
End point analysis
ABI: Real-Time PCR vs Traditional PCR (www)
Endpoint analysis
Different concentrations give similar
endpoint results!
Real-time Principles
•based on the detection and quantitation of
a fluorescent reporter
•In stead of measuring the endpoint we focus on the first
significant increase in the amount of PCR product.
• The time of the increase correlates inversely to the initial
amount of DNA template
Polymerization
5
3
Forward
Primer
R
Probe
R = Reporter
Q  Q = Quencher
3
5
5
Reverse
Primer
3
5
For Real Time PCR we need a
a specific probe with a
fluorescent reporter.
R
Probe
Q
When in close contact with the reporter,
the quencer absobes its emission.
Strand Displacement
R
5
3
5
Q
3
5
3
5
Cleavage
R
5
Q
3
3
5
5
3
5
Polymerization Completed
R
Q
3
5
3
5
5
3
Endpoint analysis
Different concentrations give similar
endpoint results!
Van der Velden. Leukemia 2003 (www)
SYBR Green
(double-stranded DNA binding dye)
* emits a strong fluorescent signal upon binding to
double-stranded DNA
* nonspecific binding is a disadvantage
* requires extensive optimisation
•longer amplicons create a stronger signal
• It´s cheap
SYBR® Green I Chemistry
Polymerization
5'
Forward
Primer
3'
5'
5'
3'
Reverse
Primer
5'
Polymerization completed
5'
3'
5'
5'
3'
5'
Real-time PCR advantages
* not influenced by non-specific amplification
* amplification can be monitored real-time
* no post-PCR processing of products
(high throughput, low contamination risk)
* requirement of 1000-fold less RNA than conventional assays
(3 picogram = one genome equivalent)
* most specific, sensitive and reproducible
Real-time PCR disadvantages
* setting up requires high technical skill and support
* high equipment cost
* Runs are more expensive than conventional PCR
* DNA contamination (in mRNA analysis)
Data analysis
Cycle Threshold
* cycle threshold or the CT value is the cycle at which
a significant increase in DRn is first detected
* it is the parameter used for quantitation
* CT value of 40 or more means no amplification and
cannot be included in the calculations
Van der Velden. Leukemia 2003 (www)
(www)
(www)
Housekeeping gene
• Knowing the amount of mRNA in one sample from one
specific gene does not tell us alot
• You don´t know the total amount of mRNA in your sample
• You also dont know how much the mRNA level has
changed compared to other mRNA levels
Example:
mRNA levels increase 2x after induction
It is possable that all genexpression in the cell has increased
We have to compare the expression of our gene to another
gene which expression is normally constant, a
housekeeping gene
Multiplexing
* TaqMan: different dyes for each target (FAM, TET,
VIC and JOE)
* SYBR green: different melting points for each target
* extensive optimisation is required
* one-step PCR cannot be used
Pure Dyes
500nm
660nm
Wavelength (nm)
What is Multiplexing?
Real-Time PCR Applications
* quantitation of gene expression
* drug therapy efficacy / drug monitoring
* viral quantitation
* pathogen detection

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