1. Real-Time PCR (Quantitative PCR)

2. Goals
Understand the fundamental difference between qPCR and traditional PCR
Understand the basic quantification method using of qPCR
Understand differences between qPCR and Northern blotting

3. Applications of real-time PCR
Powerful and reliable quantitative method
Gene expression
Determination/monitoring of viral load
Quantification of cancer genes
Microarray verification
Transgenic copy
SNP analysis

4. Steps of real-time PCR Three phases Plateau phase
Amount of PCR product
Linear phase
Exponential phase
PCR cycle number

5. Exponential amplification of PCR
Xn = X0 * (1 + E) n
E = [10(–1/slope)] – 1
(Efficiency = 1 during exponential amplification)
Xn = DNA copies at cycle n
X0 = DNA copies at cycle 0
E = efficiency of amplification
n = cycle number

6. Quantitative detection system
Fluorescence detection system
Two types of fluorochromes
DNA binding dye
Probe-based fluorochromes

7. SYBR green (DNA binding dye)
Most commonly used

8. Probe-based fluorochromes (FAM, VIC, TET, FRET)
Less commonly used now
Fluorophore
Quencher

9. SYBR green
Vs.
Probe-based Fluoro.
Does not discriminate between the gene of interest and other DNAs (i.e. contamination)
Does not allow to do multiplex PCR
Requires less steps
Less costly
Does discriminate, more specific
Allows multiplex PCR with usage of different fluoro.
Requires multiple steps
More costly

10. Detection zones qPCR vs PCR
Traditional PCR with
EtBr
Amount of PCR product
PCR cycle number
qPCR

11. Amplicon quantification by qPCR
Fluorescence increase is proportional to DNA amplification
The first cycle at which the instrument can distinguish the amplified fluorescence as being above the background level is called the threshold cycle or “Ct”

12. The threshold cycle (Ct)
Example of a Ct curve Ct

13. The threshold cycle (Ct)
Ct curves of three different samples.

14. The threshold cycle (Ct)
The Ct value is inversely proportional to the starting concentration of the sample
i.e. the greater the amount of DNA in the sample the lower the Ct value

15. Quantification methods
Absolute quantification
To determine exact amounts of DNA (e.g. viral load)
Relative quantification
To determine changes in gene expression

16. Absolute quantification
If initial amount of DNA copies is known:
XT = X0 * (1 + E) Ct
If not, Ct values of the samples has to be compared to a standard curve
XT = DNA copies at threshold
X0 = DNA copies at cycle 0
E = efficiency of amplification
Ct = threshold cycle

17. Absolute quantification
Sample of Mel1 gene which had a Ct of 22.5 cycles after amplification. What is the concentration of your amplicon?

18. Absolute quantification
Concentration of Mel1 amplicon with Ct of 22.5
y = x
22.5 = x
x =
(inverse Log 10)
The DNA concentration is µg/ml

19. Relative quantification
Normalization of the gene of interest to a housekeeping gene
Sample
Housekeeping
Ratio =

20. Real-time PCR Vs. Northern blotting More sensitive (need ~50 ng)
More accurate (can determine numbers)
DNA template (stable)
Doesn’t give size of transcripts
Faster (few hours)
Requires less steps
Less costly
Less sensitive (need ~10 ug)
Less accurate (cannot determine copy numbers)
RNA template (unstable)
Gives size of transcripts
Long (hours to days)
Requires numerous processing steps
More costly