Realtime PCR Application and Gene Analyzing

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Real-time PCR Application and Gene Analyzing
Su Ürünlerinde Uygulamali Moleküler Biyoloji
Teknikleri, 9-14 June 2008, Erzurum, Turkey

• Gen Kaneko
• Laboratory of Marine Biochemistry
• Department of Aquatic Bioscience
• Graduate School of Agricultural and Life Sciences
• The University of Tokyo

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Contents

• Introduction to real-time PCR
• Detection mechanism (TaqMan probe and SYBR
  green)
• Internal control gene
• Experimental procedure
• Data analysis

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Polymerace chain reaction (PCR)
Heat denature
30 - 40 cycles
Annealing
Extension
One PCR cycle doubles the amount of the target
DNA.
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Quantification of DNA by PCR
Fish of different size
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PCR has the limit plateau
x 220
Small amount of target mRNA in 1 µg of total RNA
1 ng
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PCR has the limit plateau
Plateau
(Introduction to quantitative RT-PCR.
Stratagene, 2007)
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DNA should be quantified before plateau
?
Electrophoresis
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Electrophoresis is one of the methods to estimate
the plateau cycle number
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Plateau
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Contents

• Introduction to real-time PCR
• Detection mechanism (TaqMan probe and SYBR
  green)
• Internal control gene
• Experimental procedure
• Data analysis

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TaqMan probe detection mechanism
Suppression of fluorescence
F
Primer
Reporter
Quencher
Degradation of TaqMan probe by DNA polymerase
Primer
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SYBR green detection mechanism
(Itoi et al., 2005)
(Introduction to quantitative RT-PCR.
Stratagene, 2007)
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Contents

• Introduction to real-time PCR
• Detection mechanism (TaqMan probe and SYBR
  green)
• Internal control gene
• Experimental procedure
• Data analysis

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Internal control gene (1)
How can we adjust their mRNA levels?
Much mRNA
Less mRNA
You cannot compare the mRNA levels of genes of
interest.
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Internal control gene (2)
Relative copy number of internal control genes is
considered to be constant.
Much mRNA of internal control gene
Less mRNA of internal control gene
Thus, mRNA levels of target gene from different
tissues can be compared by assuming the amount of
internal control gene as the same level.
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Contents

• Introduction to real-time PCR
• Detection mechanism (TaqMan probe and SYBR
  green)
• Internal control gene
• Experimental procedure
• Data analysis

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Experimental design
2 x 2 x 3 12
12 2 NTCs 14 reactions
No template controls including no RNA or cDNA
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How to make reaction mixtures

• PCR reagents
• Primers for target gene
• Without RNA

• PCR reagents
• Primers for internal control gene
• Without RNA

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Sample layout
RNAs
NTC
Primers
Target
Internal control

• RNA
• Reaction mixture

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Reagent mixture for target gene
1 reaction
8 reactions
Total
22 µL
176 µL
3 µL of RNA will be added to each mixture later.
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Contents

• Introduction to real-time PCR
• Detection mechanism (TaqMan probe and SYBR
  green)
• Internal control gene
• Experimental procedure
• Data analysis

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Results of real-time PCR is provided as the Ct
values
Ct value
(Introduction to quantitative RT-PCR.
Stratagene, 2007)
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What is Ct value?
Threshold line
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4
2
Sample A
Sample B
Sample C
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What is Ct value?
Ct value is the cycle number at which the amount
of amplified DNA reaches the threshold level.

• Ct of gene A in sample X is 25
• Ct of gene A in sample Y is 26

In sample Y, one more cycle was needed till the
amplified DNA reached the threshold level.
The mRNA level of gene A sample X sample Y 2
1
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Sample layout
cDNAs
NTC
A
B
Primers
Target
Internal control
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Comparative Ct method
Ct values of the internal control gene
Both samples contain equal amount of total RNA
Ct values of the target gene
The mRNA level of the target gene sample A
sample B 1 4
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Comparative Ct method
The relative amount of the target mRNA in sample
is
2-dCt
dCt Ct (target gene) -Ct (internal control gene)
Sample A dCt 30.0 - 22.0 8 Sample B dCt
28.0 - 22.0 6
A B 2-8 2-6 1 4
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Summary (1)

• Introduction to real-time PCR
• Detection mechanism (TaqMan probe and SYBR
  green)
• Internal control gene
• Experimental procedure
• Data analysis

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Summary (2)
Real-time PCR is useful when...

• the amount of RNA is low
• accurate quantification is necessary
• the reliable internal control gene can be used

Northern blot provides us...

• the rough estimation of mRNA amount
• high cost performance

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Other topics

• Amplification efficiency of primers
• Dissociation curve analysis
• Reference dye
• Absolute quantification