Quantification of RNA by real-time PCR

Vilborg Matre

Overview

Gene expression profile- to characterize how a cell/animal responds to a stimulus

Real-time PCR

Replaces Northerns

Conventional PCR

94oC

72oC

55oCAnnealing

Elongation

Denaturation

One cycle

= a cyclic process leading to exponentialaccumulation of a specific DNA- small amounts of DNA can be detected- Nobel priced method

time

Conventional PCR versus real-time PCR

Conventional PCR- end-point method- detection after PCR

Real-time PCR- continuous measurement- log-phase quantitation

N : number of amplified moleculesn : number of amplification cycles

high concentration /high efficiency

high concentration /low efficiency

low concentration /high efficiency

N

n

end-point analysislog-phase analysis

Conventional PCR versus real-time PCR

Fluorescence - the clue to real-time PCR

Fluorescence!

Detection of PCR-product while formed via fluorescense

Alternative I: SYBR-greenmeasuring accumulated total DNA

Alternative II: Hybridization-probesmeasuring accumulated specific DNA

Theoretical aspects

PCR-basis: N = NN00 x 2 x 2nn

– N: number of amplified molecules

– N0: initial number of molecules

– n: number of amplification cycles

exponential

N

n

Curve

Theoretical aspects

Log-transformation:a linear curve for each reaction

linear

Log N

n

Formula

Log N = log N0 + n log2

Starting amount

The accumulation of PCR product can be fully describedby this linear curve, and only two points are necessary to describe it

N: number of amplified molecules n: number of amplification cyclesN0: initial number of molecules E: amplification efficiency

Real

Theory

PCR QuantificationTheoretical and practical aspects

N = N0 x (Econst)n

N = N0 x 2n

N = N0 x (Evar)n

N

n

N

n

N

N0

N

N0

end-point-PCR

log-phase-PCR

Automatic quantification by the Lightcycler

• Unknown Sample

• Standard Curve

log

(F2

/F1

)

Target

n

log

(F2

/F1

)

Cro

ssin

g P

oin

t (C

ycl

es)

log (copy number)

Standard Curve

Unknown Sample

Quantification: Concept for the LightCycler

Cro

ssin

g P

oin

t (C

ycl

es)

log (copy number)n

log

(F2

/F1

)

n

log

(F2

/F1

)

Housekeeping gene

n

log

(F2

/F1

)

Target

Fluorescence detection- an example

with E = 1.9Fluorescence detectedwhen N = 1010 copies!

Cycle n = 14N0=106

N = NN00 x E x Enn

Fluorescence detection- an example

with E = 1.9Fluorescence detectedwhen N = 1010 copies!

Cycle n = 25Cycle n = 36

N0=103

N0=1

N = NN00 x E x Enn

LightCyclerQuantification - what it looks like

Template: Plasmid; Target: CycA; Detection Format: Hybridization Probes

Standard Calculated concentration

9.522E+91.024E+99.433E+71.127E+71.029E+69.902E+41.021E+49.217E+29.276E+11.085E+1H2O

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+101.0E+91.0E+81.0E+71.0E+61.0E+51.0E+41.0E+31.0E+21.0E+1H2O

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

1.0E+101.0E+91.0E+81.0E+71.0E+61.0E+51.0E+41.0E+31.0E+21.0E+1H2O

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

= 1.97

293Tet-Off/Dox+/AMV = 0.0351

293Tet-Off/Dox+/PP1

293Tet-Off/Dox-/AMV = 0.1907

293Tet-Off/Dox-/PP1

5.4 fold up

Another experiment

• Evaluation Parameters– Error = 0.142

– r = - 1.00

– Slope

• Melting curve analysis– Primer dimers

– Expected melting point

• Calculations

E = 10 -1/slope

E = 10 -1/-3.475

= 1.94

293Tet-Off/Dox+/AMV = 0.0168

293Tet-Off/Dox+/PP1

293Tet-Off/Dox-/AMV = 0.0871

293Tet-Off/Dox-/PP1

5.2 fold up

Additional informationMelting 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 ofMelting 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