Real-time PCR: Introduction and definition.

1. q Real-Time PCR
Ahmed Housanie El-Sappah
Teacher of genetics
Genetics Department
ahmed_elsappah2006@yahoo.com

2. Objectives
This presentation will cover the following
topics:
1. What is Real Time- PCR?
2. How does real-time PCR work?
3. Real time- PCR Vs. Traditional PCR
4. Real time- PCR instruments?
5. Detection Chemistry
6. Real time- PCR data analysis
- Example : Determining expression of Cytochrome
p450 1A (CYP1A) in Nile tilapia

3. Definition
History
How it Works ?
Different with traditional PCR

4. What is Real Time- PCR ?
Nomenclature
(Kinetic PCR ) Kinetic polymerase chain reaction
(qPCR) Quantitative polymerase chain reaction
(qRT PCR ) Quantitative Real-Time polymerase chain reaction
Not
(RT-PCR) Reverse transcriptase PCR
Definition
A real-time polymerase chain reaction is a laboratory
technique of molecular biology based on
the polymerase chain reaction (PCR). It monitors
the amplification of a targeted DNA molecule during
the PCR, i.e. in real-time,and not at its end

5. History of Real time - PCR

6. How does real-time PCR work?
To best understand what
real-time PCR is, let’s review h
ow regular PCR works……?

7. The Polymerase Chain Reaction
5’
5’
3’
3’
d.NTPs
Thermal Stable
DNA Polymerase
Primers
Denaturation
Annealing
Add to Reaction Tube
Samples
DNA
DNA

8. Extension
5’ 3’
5’
3’
Extension Continued
5’ 3’
5’
3’
Taq
Taq
3’
5’
3’
Taq
Taq
Repeat

9. 5’
3’
3’
3’
3’
5’
3’
3’
5’
3’
3’
Cycle 2
4 Copies
Cycle 3
8 Copies
3’
3’
5’
3’
3’
5’
3’
3’
5’
3’
3’
5’
3’
3’
5’
3’
3’
5’
3’
3’
5’
3’
3’

10. Samples
10
DNA RNA
q RT-PCR
Two ways
One step Two steps
cDNA
q RT-PCR
q RT-PCR
Gene specific
primers
Gene specific
primers
Oligo ( dt)

13. Real time- PCR Vs. Traditional PCR

14. Real-Time PCR PCR
Sensitivity High Low
Specificity High
-use specific probes
Low
-only size discrimination
Quantitative
results
Yes
-Specific fluorescence
No
-EtBr staining
Detection
method
Probe-specific
Fluorescence
Agarose gel
Electrophoresis
Detection range Wide range Short range (<2 log)
Reaction time 1 hr 3-5 hr
Post-PCR steps No Agarose gel electrophoresis

15. What Type of Instruments are
used with Real-Time PCR?
Real-time PCR instruments consist of THREE
main components:
1. Thermal Cycler .
2. Optical Module (detect fluorescence in the tubes during the run)
3. Computer (to translate the fluorescence data into meaningful
results)
1
2
3

16. Instrument available
instrument Light source No. of
sample
ِABI prism 7900HT
Argon laser
(500-660nm)
96 - 384
ABI prism 7000 SDS Tungsten-
halogen
96
Applied Biosystem 7300
Single-excitation
, four-emission
filters and CCD c
amera
96
Applied Biosystem 7500 Five-excitation ,
five-emission
filters and CCD c
amera
96
Light cycler2.0 (roche)
Blue led (470nm) 30

17. I Cycler iQ (BioRad
Instrument)
Tungsten – halogen
lamp (400-700 nm)
96 - 384
Mx3000 p
(stratagene)
Quartz-tungsten
halogen
(350-750 nm)
96
Mx4000 Multiplex
(stratagene)
Quartz-tungsten
halogen
(350-750 nm)
96
Smart cycler
systems 1600-9600
( Cepheid)
Four channel
(450-495 , 500-550 ,
565-590 and 630-650)
16- 96
Rotor Gene 3000
Or 6000
(470 , 530 ,585 and
625 nm)
36-72

18. Detection Chemistry

19. Primer
- Short (Often < 50 nt) oligonucleotide sequence of
DNA
- Complementary to the beginning and the end of the
target DNA sequence
- Needed to initiate the synthesis of new DNA in a
PCR reaction
- Involved in AMPLIFICATION
Probe
- A single-stranded DNA with a specific base
sequence
- Labeled with fluorescence dyes (TaqMan probe)
- Used to detect the complementary base sequence of
target DNA/RNA by hybridization
- Involved in DETECTION
Reporter dye / Quencher dye
Primer & Probe

20. Detection types
1) Non- specific fluorescent dye
Sybr green .
2) Sequence –specific DNA probes
Hydrolysis .
Hybridization .

21. 2) Annealing
F
F
F
F
F
F
F
F
F
F
F
F
1) Denaturation
3) Extension
1) Intercalating dye fluoresces
more brightly when bound to
dsDNA.
2) DNA binding dyes are
inexpensive compared to the
other probes.
3) SYBR Green I, EtBr
Intercalating method
SYBR Green dye
Asymmetrical cyanine dye
C32H37N4S+

22. Hydrolysis Probe
Fluorescent
reporter dye at the
5’ end is quenched
by fluorescent
quencher dye at the
3’ end

23. Hydrolysis types :-
1- Taq Man probe .
2- Beacon probe .
3- Scorpions probe .

24. 1) Fluorescent reporter dye at the 5’
end is quenched by fluorescent
quencher dye at the 3’ end.
2) When amplification occurs the
TaqMan probe is degraded due
to the 5'-->3' exonuclease activity
of Taq DNA polymerase, thereby
separating the quencher from the
reporter during extension.
3) The TaqMan assay accumulates
a fluorescence signal.
2) Annealing
1) Denaturation
3) Extension
Q
R
R Q
Taq
R
Q
R Q
TaqMan® probe
1- TaqMan Probe

25. 1) A molecular beacon begins as a
stem-and-loop structure. The
sequences at the ends of the
probe match and bind, creating
the stem
2) When the probe binds to a single-
stranded DNA template, the
structure unfolds, separating the
quencher from the dye and
allowing fluorescence.
2) Annealing
1) Denaturation
3) Extension
Q
R
Q
Taq R
Q
R
Molecular Beacon
2- Molecular Beacon Probe

26. 3- Scorpion
Probe

27. 1) FRET method designed two
specifically probe. It labeled with
different dyes, such as at the 5’
end of donor probe and at the 3’
end of acceptor probe.
2) At close proximity, the donor dye
(D)is excited by the light source
and the energy is transferred the
acceptor dye(R). Subsequently,
fluorescent light is emitted at a
different wavelength.
2) Annealing
1) Denaturation
3) Extension
D R
Taq
D R
D R
Energy
transfer
Hybridization Probe (FRET)
Hybridization Probe

28. Analysis of data

30. Analysis of data
According to experimental goals and
available resources
there are two types of RT-PCR analysis:-
1- Absolute quantification .
2- Relative quantification .

31. 1- Absolute Quantification
Understand properties intrinsic to a given
sample
Example
-chromosome or gene copy number
determination .
-Viral load measurement .
Doesnot give direct answer about changes of
difference in gene expression

32. 2- Relative Quantification
Compare level or changes in gene expression .
Example
-Applicable to multiple gene
-To perform relative quantification you need
a calibrator sample .
There are two types of relative analysis:-
a- Normalization a gainst unit mass.
b-Normlization against refrence gene

33. a- Normalization a gainst unit mass
- Uses a mass as calibrator.
- Accurate quantification of starting material.
- One sample chosen as calibrator.
- Expression of target gene in all samples
increase or decrease relative calibration.

34. Normalization a gainst unit mass
Ratio (test/calibrate)= 2 ct calibrate- ct test
Normal equation
Ratio (test/calibrate)= 2 ∆ct
Where
∆ct= Ct calibrate – Ct test

35. Example
Examine relative expression of CYP1A gene in
2 samples
(Control and treatment)
C CT = 15.0
T CT = 12.0
Ratio (test/calibrate)= 2 ∆ct
Where
∆ct= Ct calibrate – Ct test = 15-12= 3
Ratio= 23 = 8
This mean that the expression of
CYP1A gene is 8 folds higher than Control.

36. b-Normlization against refrence gene
- Require known reference gene with
stable expression level
Note that
(housekeeping genes)
are typically constitutive genes that are required for
the maintenance of basic cellular function, and
are expressed in all cells of an organism under
normal and patho-physiological conditions

37. Normlization against refrence gene:-
1) Livak (∆∆ct ) method.
2) ∆ct method using reference gene .
3)Pfaffi method.

38. CT
CYP1A
CT
ß-Actine
∆ct ∆∆ct=
∆ctt - ∆ctc
2(- ∆∆ct)
C 15.0 16.5 -1.5 0
1
T 12.0 15.9 -3.9 -2.4
5.3
1) Livak (∆∆ct ) method.

39. CT
ß-Actine
CT
CYP1A
∆c
t 2∆ct
Normalize
Expression
Expression=
Any 2∆ct/ 2∆ctC
C 16.5 15.0 1.5 2.83
1
T 15.9 12.0 3.9 14.93
5.3
2) ∆ct method using reference gene

40. What is Real-Time PCR used for?
Real-Time PCR has become a cornerstone
of molecular biology:
• Gene expression analysis
– Cancer research
– Drug research
• Disease diagnosis and management
– Viral quantification
• Food testing
– Percent GMO food
• Animal and plant breeding
– Gene copy number

41. Example

42. Example
Determining expression of Cytochrome
p450 1A (CYP1A) in Nile tilapia
(CYP1A) associated with detoxification of organic
compounds such as PAH (polyaromatic hydrocarbons)

43. Method
1- Isolation of RNA from tissue
Note: no contamination
From liver and gill under
control and treatment
Fish Fish
Liver Gill Liver Gill
c c T T

44. 2- Reverse transcriptase to cDNA
5 RNA +d water
cD
NA
45°c 60 min
95°c 5 min
cDNA
c c T T

45. 3- Real time PCR
Control Treatment
Liver Gill Liver Gill
cDNA cDNA cDNA cDNA
CYP1A
F 5‘- AGTTTGTTGCTCCTGCTGTATTGTT- 3‘
R 5‘ GGTTGGATAGTTTAGGATGCTGCA -3
ß-ACTINE
F- 5‘-GGGTCAGAAAGACAGCTACGTT - 3‘
R- 5‘-CTCAGCTCGTTGTAGAAGGTGT -3‘
cDNA
SYBR Green
Specific primer

48. 4- analysis
Sample
name
Averge CT
CYP1A
Averge CT
B-actin
∆ct ∆∆ct 2(- ∆∆ct)
Liver
conrol
25.06 23.42 1.63 0
1
Liver
test
15.49 20.85 -5.36 -6.99
127.1
Gill
conrol
30.75 22.13 8.62 0
1
Gill
test
25.24 21.33 3.92 -4.7
26

49. Tissue 2(- ∆∆ct)
Liver
conrol
1
Liver
test
127.1
Gill
conrol
1
Gill
test
26
0
20
40
60
80
100
120
140
Liverc
Gillc
Livert
Gillt

50. References
Abeer, A. I., Y. Kaminishi, M. M. Osman, Z . H. Abdel Wahad , M.A. El-
Kady and T. Itakura (2009). Development and application of
a real-time quantitative PCR assay for determining expression of
benzo-a-pyrene-inducible cytochrome P450 1A in Nile tilapia . Afr
ican
journal of biotechnology vol 8 (23) pp. 6588-6595.
Livak, J. and Thomas D. S. (2001). Analysis of relative Gene
Expression Data Using Real-Time Quantitative PCR and the
2(- ∆∆ct) method . Elsevier Science (USA) 1046-2023.
Mariza , L. and J.F. Medrano (2005). Real-time PCR for mRNA quantit
ation Biotechniques University of California ,Davis CA , USA Vol.39,
No 1
STRATAGENE (2004) Introduction to Quantitative PCR ,Methods and A
pplication Guide.In #70200-00/Revision #084001.

51. Thank you