Quantification of RNA by real-time
PCR
Vilborg Matre
Overview
Real-time PCR
Replaces Northerns
Gene expression profile
- to characterize how a cell/animal
responds to a stimulus
Conventional PCR
One cycle
Denaturation
94oC
72oC
Elongation
= a cyclic process
leading to exponential
accumulation of
a specific DNA
- small amounts of DNA
can be detected
- Nobel priced method
55oC
Annealing
time
Conventional PCR
versus real-time PCR
Conventional PCR
- end-point method
- detection after PCR
Real-time PCR
- continuous measurement
- log-phase quantitation
Conventional PCR
versus real-time PCR
N
log-phase analysis
end-point analysis



n
high concentration /
high efficiency
high concentration /
low efficiency
low concentration /
high efficiency
N : number of amplified
molecules
n : number of amplification
cycles
Fluorescence
- the clue to real-time PCR
Fluorescence!
Detection of PCR-product while formed
via fluorescense
Alternative I: SYBR-green
measuring accumulated total DNA
Alternative II: Hybridization-probes
measuring accumulated specific DNA
Theoretical aspects
PCR-basis: N = N0 x 2n
– N: number of amplified molecules
– N0: initial number of molecules
– n: number of amplification cycles
N
Curve
exponential
n
Theoretical aspects
Log-transformation:
a linear curve for each reaction
Formula
Log N
linear
Log N = log N0 + n log2
Starting amount
n
The accumulation of PCR product can be fully described
by this linear curve, and only two points are necessary to describe it
PCR Quantification
Theoretical and practical aspects
N = NN0 x 2n
N
Theory
log-phase-PCR
N = N0 x (Econst)n
N0
n
N
N
Real
N0
n
N: number of amplified molecules
N0: initial number of molecules
end-point-PCR
N = N0 x (Evar)n
n: number of amplification cycles
E: amplification efficiency
Automatic quantification by the
Lightcycler
Unknown
Sample
•
Standard
Curve
Target
log (F2/F1)
Crossing Point (Cycles)
log (F2/F1)
•
n
log (copy number)
Quantification: Concept for the LightCycler
log (F2/F1)
Unknown Sample
Housekeeping gene
log (F2/F1)
Target
log (F2/F1)
Standard Curve
n
Crossing Point (Cycles)
n
n
log (copy number)
Fluorescence detection
- an example
N = N0 x En
N0=106
with E = 1.9
Fluorescence detected
when N = 1010 copies!
Cycle n = 14
Fluorescence detection
- an example
N = N0 x En
N0=103
N0=1
with E = 1.9
Fluorescence detected
when N = 1010 copies!
Cycle n = 25
Cycle n = 36
LightCycler
Quantification - what it looks like
Standard
1.0E+10
1.0E+10
1.0E+10
1.0E+10
1.0E+9
1.0E+9
1.0E+9
1.0E+9
1.0E+8
1.0E+8
1.0E+8
1.0E+8
1.0E+7
1.0E+7
1.0E+7
1.0E+7
1.0E+6
1.0E+6
1.0E+6
1.0E+6
1.0E+5
1.0E+5
1.0E+5
1.0E+5
1.0E+4
1.0E+4
1.0E+4
1.0E+4
1.0E+3
1.0E+3
1.0E+3
1.0E+3
1.0E+2
1.0E+2
1.0E+2
1.0E+2
1.0E+1
1.0E+1
1.0E+1
1.0E+1
HH
H2O22OO
Calculated
concentration
9.522E+9
1.024E+9
9.433E+7
1.127E+7
1.029E+6
9.902E+4
1.021E+4
9.217E+2
9.276E+1
1.085E+1
H2 O
Template: Plasmid; Target: CycA; Detection Format: Hybridization Probes
Interpretations of the results
• Evaluation Parameters
– Error < 1
– r = -1.00
– Slope
E = 10 -1/slope
E = 10 -1/-3.407
= 1.97
• Melting curve analysis
– Primer dimers
– Expected melting point
• Calculations
293Tet-Off/Dox+/AMV
293Tet-Off/Dox+/PP1
= 0.0351
5.4 fold up
293Tet-Off/Dox-/AMV
293Tet-Off/Dox-/PP1
= 0.1907
Another experiment
• Evaluation Parameters
– Error = 0.142
– r = - 1.00
– Slope
E = 10 -1/slope
E = 10 -1/-3.475
= 1.94
• Melting curve analysis
– Primer dimers
– Expected melting point
• Calculations
293Tet-Off/Dox+/AMV
293Tet-Off/Dox+/PP1
= 0.0168
5.2 fold up
293Tet-Off/Dox-/AMV
293Tet-Off/Dox-/PP1
= 0.0871
Additional information
Melting curve analysis
• AFTER amplification - the Lightcycler can perform
a second type of analysis: precise determination of
the melting point (Tm) of the product
• Procedure
– After the PCR run the temperature is slowly raised while the
fluorescence is measured. As soon as the dsDNA starts to
denature, the SYBR green dye is released, resulting in
decrease in fluorescence.
Benefits of
Melting curve analysis
• Confirmation of PCR product identity
– Each product has its specific Tm
– One peak - one product, several peaks - many products
• Differentiation of specific PCR product from nonspecific products
– such as primer dimers