UNIVERSITAS

GADJAH MADA

Quantitative Real-Time
PCR (qPCR)

dr. Ahmad Hamim Sadewo, Ph.D

KURSUS
BIOLOGI MOLEKULER & IMUNOLOGI
S2 IlmuKedokteran Tropis
Fakultas Kedokteran Universitas Gadjah Mada
14 - 19 Maret 2016
 Information from mRNA levels

DNAmRNAprotein

• Reflect level of gene expression

• Information about cell response
• Protein production
 Types of RNA/DNA analysis

Direct
-Northern blotting
-In situ hybridization

PCR amplification
-Regular RT-PCR  band’s density
-Real time PCR
(Microarrays)
 Nomenclature

RT-PCR = Reverse Transcriptase PCR

qRT-PCR = quantitative Real-Time PCR

or simply qPCR
• NEED TO QUANTITATE DIFFERENCES
IN mRNA EXPRESSION

• SMALL AMOUNTS OF mRNA

– LASER CAPTURE
– SMALL AMOUNTS OF TISSUE
– PRIMARY CELLS
– PRECIOUS REAGENTS
5
• QUANTITATION OF mRNA
– northern blotting
– ribonuclease protection assay
– in situ hybridization
– PCR
• most sensitive
• can discriminate closely related mRNAs
• technically simple
• but difficult to get truly quantitative results using
conventional PCR

6
 Steps in qRT-PCR Methods to
Analyze Gene Expression

• Isolate RNA
• cDNA synthesis (RT-PCR)
• PCR reaction
Why isn´t this good enough?
Weakness of Agarose
Gels

* Low sensitivity
* Low resolution
* Non-automated
* Size-based discrimination only
* Results are not expressed as numbers
 based on personal evaluation
• Ethidium bromide staining is not very
quantitative
• End point analysis
ABI: Real-Time PCR vs Traditional PCR (www)
 Endpoint analysis

Different concentrations give similar

endpoint results
 Real-time Principles

•based on the detection and quantitation of

a fluorescent reporter

•In stead of measuring the endpoint we focus

on the first significant increase in the amount
of PCR product.

• The time of the increase correlates

inversely to the initial amount of DNA
template
CYCLE NUMBER AMOUNT OF DNA
0 1
1 2
2 4
3 8
4 16
5 32
6 64
7 128
8 256
9 512
10 1,024
11 2,048
12 4,096
13 8,192
14 16,384
15 32,768
16 65,536
17 131,072
18 262,144
19 524,288
20 1,048,576
21 2,097,152
22 4,194,304
23 8,388,608
24 16,777,216
25 33,554,432
26 67,108,864
27 134,217,728
28 268,435,456
29 536,870,912
30 1,073,741,824
31 1,400,000,000
32 1,500,000,000 12
33 1,550,000,000
34 1,580,000,000
CYCLE NUMBER AMOUNT OF DNA
0 1
1 2
2 4 1600000000
3 8
1400000000
4 16

AMOUNT OF DNA
5 32 1200000000
6 64 1000000000
7 128
800000000
8 256
9 512 600000000
10 1,024 400000000
11 2,048 200000000
12 4,096
13 8,192 0
14 16,384 0 5 10 15 20 25 30 35
15 32,768 PCR CYCLE NUMBER
16 65,536
17 131,072
18 262,144
19 524,288
20 1,048,576 10000000000
21 2,097,152 1000000000
22 4,194,304
AMOUNT OF DNA

100000000
23 8,388,608 10000000
24 16,777,216 1000000
25 33,554,432 100000
26 67,108,864 10000
27 134,217,728
1000
28 268,435,456
100
29 536,870,912
10
30 1,073,741,824
1
31 1,400,000,000
32 1,500,000,000 0 5 10 15 20 25 30 35
13
33 1,550,000,000 PCR CYCLE NUMBER
34 1,580,000,000
 1600000000 1600000000
1400000000 1400000000

AMOUNT OF DNA

AMOUNT OF DNA
1200000000 1200000000
1000000000 1000000000
800000000 800000000
600000000 600000000
400000000 400000000
200000000 200000000
0 0
0 5 0 10 5 15 10 20 15 25 20 30 25 35 30 35
PCR CYCLE NUMBER
PCR CYCLE NUMBER

14
15
16
 10000000000 10000000000
1000000000 1000000000

AMOUNT OF DNA

AMOUNT OF DNA
100000000 100000000
10000000 10000000
1000000 1000000
100000 100000
10000 10000
1000 1000
100 100
10 10
1 1
0 5 010 515 1020 1525 2030 2535 30 35
PCR CYCLE NUMBER
PCR CYCLE NUMBER

17
 REAL TIME PCR
• kinetic approach
• early stages
• while still linear

18
www.biorad.com
 SYBR Green/Evagreen
(double-stranded DNA binding dye)

* emits a strong fluorescent signal upon

binding to double-stranded DNA
* nonspecific binding is a disadvantage
* requires extensive optimisation
•longer amplicons create a stronger signal
• It´s cheap
 Chemistry Reaction

Polymerization
Forward
Primer
5'
3' 5'

5' 3'
5'
Reverse
Primer

Polymerization completed
5'
3' 5'

5' 3'
5'
 Real-time PCR advantages

* not influenced by non-specific amplification

* amplification can be monitored real-time
* no post-PCR processing of products
(high throughput, low contamination risk)
* requirement of 1000-fold less RNA than
conventional assays
(3 picogram = one genome equivalent)
* most specific, sensitive and reproducible
 Real-time PCR disadvantages

* setting up requires high technical skill and

support
* high equipment cost
* Runs are more expensive than conventional
PCR

* DNA contamination (in mRNA analysis)

 Data analysis
Cycle Threshold

* cycle threshold or the CT value is the cycle at which

a significant increase in DRn is first detected
* it is the parameter used for quantitation
* CT value of 40 or more means no amplification and
cannot be included in the calculations
Van der Velden. Leukemia 2003 (www)
(www)
 Standards
• same copy number in all cells
• expressed in all cells
• medium copy number advantageous
– correction more accurate

27
 Standards
• Commonly used standards
– Glyceraldehyde-3-phosphate dehydrogenase mRNA
– Beta-actin mRNA
– MHC I (major histocompatability complex I) mRNA
– Cyclophilin mRNA
– mRNAs for certain ribosomal proteins
• E.g. RPLP0 (ribosomal protein, large, P0; also known as
36B4, P0, L10E, RPPO, PRLP0, 60S acidic ribosomal
protein P0, ribosomal protein L10, Arbp or acidic
ribosomal phosphoprotein P0)
– 28S or 18S rRNA
28
(www)
 Housekeeping gene
• Knowing the amount of mRNA in one sample from one
specific gene does not tell us alot
• You don´t know the total amount of mRNA in your sample
• You also dont know how much the mRNA level has
changed compared to other mRNA levels
Example:
mRNA levels increase 2x after induction
It is possable that all gene expression in the cell has increased
We have to compare the expression of our gene to another
gene which expression is normally constant, a
housekeeping gene
 Pure Dyes

500nm 660nm
Wavelength (nm)
 Appication of Real-Time PCR

1. quantitation of gene expression

2. Gene copy number analysis
3. drug therapy efficacy / drug monitoring
4. viral quantitation
5. pathogen detection
 Probe Analysis
Polymerization

R = Reporter
Forward
Primer R Probe Q  Q = Quencher
5 3
3 5

5 3
5
Reverse
Primer
For Real Time PCR we need a
a specific probe with a
fluorescent reporter.

R
Probe Q
When in close contact with the reporter,
the quencer absobes its emission.
 Strand Displacement

R
Q
5 3
3 5

5 3
5
 Cleavage

R
Q
5 3
3 5

5 3
5
 Polymerization Completed

R Q
3

5
3 5

5 3
 Endpoint analysis

Different concentrations give similar

endpoint results
Van der Velden. Leukemia 2003 (www)