1. Quantification of RNA by real-time PCR
Vilborg Matre

2. Overview Real-time PCR Replaces Northerns Gene expression profile
- to characterize how a cell/animal
responds to a stimulus
Replaces Northerns

3. Conventional PCR One cycle time 94oC = a cyclic process
Denaturation
94oC
= a cyclic process
leading to exponential
accumulation of
a specific DNA
- small amounts of DNA
can be detected
- Nobel priced method
72oC
Elongation
55oC
Annealing
time

4. Conventional PCR versus real-time PCR
- end-point method
- detection after PCR
Real-time PCR
- continuous measurement
- log-phase quantitation

5. Conventional PCR versus real-time PCR
log-phase analysis
end-point analysis N
high concentration /
high efficiency
low efficiency
low concentration /
Author: T. Emrich
Def
Real-time, kinetic quantification allows measurements to be made during the log-linear phase of a PCR reaction and is a fast and accurate way to quantify PCR products.
Real-time PCR can be used for quantification of RNA or DNA and for mutation detection.
N : number of amplified
molecules
n : number of amplification
cycles n

6. Fluorescence - the clue to real-time PCR

7. Detection of PCR-product while formed via fluorescense
Alternative I: SYBR-green
measuring accumulated total DNA
Alternative II: Hybridization-probes
measuring accumulated specific DNA

8. Theoretical aspects PCR-basis: N = N0 x 2n
N: number of amplified molecules
N0: initial number of molecules
n: number of amplification cycles
exponential N n
Curve

9. 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

10. Theoretical and practical aspects
PCR Quantification
Theoretical and practical aspects
N = N0 x 2n
Theory
log-phase-PCR
N = N0 x (Econst)n
Real
end-point-PCR
N = N0 x (Evar)n
Author: T. Emrich
N: number of amplified molecules n: number of amplification cycles
N0: initial number of molecules E: amplification efficiency

11. Automatic quantification by the Lightcycler
Unknown Sample
Standard Curve
Target
log (F2/F1)
log (F2/F1)
Crossing Point (Cycles)
Author: T. Emrich n
log (copy number)

12. Quantification: Concept for the LightCycler
log (F2/F1)
Target n
log (F2/F1)
Housekeeping gene
Unknown Sample n
log (F2/F1)
Crossing Point (Cycles)
log (copy number)
Standard Curve
Author: T. Emrich

13. Fluorescence detection - an example
N = N0 x En
with E = 1.9
Fluorescence detected
when N = 1010 copies!
Cycle n = 14
N0=106

14. Fluorescence detection - an example
N = N0 x En
with E = 1.9
Fluorescence detected
when N = 1010 copies!
Cycle n = 25
Cycle n = 36
N0=103
N0=1

15. LightCycler Quantification - what it looks like
Standard Calculated concentration
1.0E+10
1.0E+9
1.0E+8
1.0E+7
1.0E+6
1.0E+5
1.0E+4
1.0E+3
1.0E+2
1.0E+1
H2O
1.0E+10
1.0E+9
1.0E+8
1.0E+7
1.0E+6
1.0E+5
1.0E+4
1.0E+3
1.0E+2
1.0E+1
H2O
1.0E+10
1.0E+9
1.0E+8
1.0E+7
1.0E+6
1.0E+5
1.0E+4
1.0E+3
1.0E+2
1.0E+1
H2O
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
H2O
1.0E+10
1.0E+9
1.0E+8
1.0E+7
1.0E+6
1.0E+5
1.0E+4
1.0E+3
1.0E+2
1.0E+1
Author: I. Obermaier
Template: Plasmid; Target: CycA; Detection Format: Hybridization Probes

16. Interpretations of the results
Evaluation Parameters
Error < 1
r = -1.00
Slope
Melting curve analysis
Primer dimers
Expected melting point
Calculations
E = 10 -1/slope
E = 10 -1/-3.407 =
293Tet-Off/Dox+/AMV =
293Tet-Off/Dox+/PP1
293Tet-Off/Dox-/AMV =
293Tet-Off/Dox-/PP1
5.4 fold up

17. Another experiment Evaluation Parameters Melting curve analysis
Error = 0.142
r =
Slope
Melting curve analysis
Primer dimers
Expected melting point
Calculations
E = 10 -1/slope
E = 10 -1/-3.475 =
293Tet-Off/Dox+/AMV =
293Tet-Off/Dox+/PP1
293Tet-Off/Dox-/AMV =
293Tet-Off/Dox-/PP1
5.2 fold up

18. 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.

19. 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 non-specific products
such as primer dimers