Quantification of RNA by real-time PCR

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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 Replaces Northerns

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

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

Conventional PCR versus real-time PCR log-phase analysis end-point analysis N high concentration / high efficiency low efficiency low concentration / Author: T. Emrich - 000207 - 49 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

Fluorescence - the clue to real-time PCR

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 exponential N n Curve

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

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

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)

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

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

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

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

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 Melting curve analysis 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 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 non-specific products such as primer dimers