Transcript in real time

PCR quantitativo
What is
Real-Time
PCR?
Real-Time PCR is a specialized technique
that allows a PCR reaction to be visualized
“in real time” as the reaction progresses.
This enables researchers to quantify the
amount of DNA in the sample at the start of
the reaction!
What is
Real-Time
PCR?
Differences with normal PCR?
• 20ul PCR reactions
• SYBR Green or probes




94°C 4 min
94°C 15 sec
40x
61°C 30 sec
72°C 30 sec
What is
Real-Time
PCR used
for?
Real-Time PCR has become a cornerstone of
molecular biology:
• Gene expression analysis
– Medical research
– Drug research
• Disease diagnosis
– Viral quantification
• Food testing
– Percent GMO food
• Transgenic research
– Gene copy number
What is
Real-Time
PCR?
Taq polymerase can only
synthesize DNA, so how
do we study gene
expression (RNA) using
qPCR?
Reverse transcription
RNA -> DNA (cDNA)
What’s Wrong With
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

Endpoint analysis
Different concentrations give
similar endpoint results!
…So that’s how PCR is usually presented.
Imagining
Real-Time
PCR
To understand real-time PCR, let’s imagine
ourselves in a PCR reaction tube at cycle
number 25…
Imagining
Real-Time
PCR
What’s in our tube, at cycle number 25?
A soup of nucleotides, primers, template,
amplicons, enzyme, etc.
~1,000,000 copies of the amplicon right now.
Imagining
Real-Time
PCR
How did
we get
here?
What was it like last cycle, 24?
Almost exactly the same, except there were only
500,000 copies of the amplicon.
And the cycle before that, 23?
Almost the same, but only 250,000 copies of the
amplicon.
And what about cycle 22?
Not a whole lot different. 125,000 copies of the
amplicon.
Imagining
Real-Time
PCR
If we were to graph the amount of DNA in our
tube, from the start until right now, at cycle
25, the graph would look like this:
2000000
1800000
How did
we get
here?
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
0
5
10
15
20
25
30
35
40
Imagining
Real-Time
PCR
So where
are we
going?
What’s it going to be like after the next cycle, in cycle 26?
Probably there will be 2,000,000 amplicons.
And cycle 27?
Maybe 4,000,000 amplicons.
And at cycle 200?
In theory, there would be
1,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000
,000,000,000,000 amplicons…
?
2000000
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
0
5
10
15
20
25
30
35
40
Imagining
Real-Time
PCR
If we plot the amount of DNA in our tube going
forward from cycle 25, we see that it actually
looks like this:
5000000
4500000
So where
are we
going?
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
0
•
•
5
10
15
20
25
30
35
40
Realistically, at the chain reaction progresses, it gets
exponentially harder to find primers, and nucleotides.
And the polymerase is wearing out.
So exponential growth does not go on forever!
Imagining
Real-Time
PCR
Measuring
Quantities
How can all this be used to measure DNA
quantities??
What if YOU started with FOUR times as much
DNA template as I did?
I have 1,000,000 copies at cycle 25.
You have 4,000,000 copies!
So… You had 2,000,000 copies at cycle 24.
And… You had 1,000,000 copies at cycle 23.
Imagining
Real-Time
PCR
So… if YOU started with FOUR times as much
DNA template as I did…
Then you’d reach 1,000,000 copies exactly
TWO cycles earlier than I would!
5000000
4500000
4000000
Measuring
Quantities
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
0
5
10
15
20
25
30
35
40
Imagining
Real-Time
PCR
What if YOU started with EIGHT times LESS DNA template
than I did?
You’d only have 125,000 copies right now at cycle 25…
…and you’ll have 250,000 at 26, 500,000 at 27, and by cycle
28 you’ll have caught up with 1,000,000 copies!
So… you’d reach 1,000,000 copies exactly THREE cycles
later than I would!
5000000
4500000
Measuring
Quantities
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
0
5
10
15
20
25
30
35
40
The “ct value”
Imagining
Real-Time
PCR
•
•
The value that represents the cycle number where the
amplification curve crosses an arbitrary threshold.
Ct values are directly related to the starting quantity of
DNA, by way of the formula:
Quantity = 2^Ct
5000000
Ct Values:
4500000
Measuring
Quantities
4000000
3500000
25
23
3000000
28
2500000
Threshold
2000000
1500000
1000000
500000
0
0
5
10
15
20
25
30
35
40
threshold
Ct
Imagining
Real-Time
PCR
There’s a DIRECT relationship between the
starting amount of DNA, and the cycle number
that you’ll reach an arbitrary number of DNA
copies (Ct value).
DNA amount = 2 ^ Cycle Number
C o p y N u m b e r v s. C t - St a n d a r d C u r v e
40
35
y =
R
25
Ct
Measuring
Quantities
30
-3 . 3 1 9 2 x +
2
=
3 9 .77 2
0 .9 9 6 7
20
15
10
5
0
0
1
2
3
4
5
6
7
L o g o f c o p y n u m b e r (1 0 n )
8
9
10
11
How do
We
Measure
DNA in a
PCR
Reaction?
We use reagents that fluoresce in the
presence of amplified DNA!
Ex. SYBR Green dye
They bind to double-stranded DNA and
emit light when illuminated with a
specific wavelength.
SYBRgreen
How do
We
Measure
DNA in a
PCR
Reaction?
• dsDNA intercalating dye
• Unspecific (optimization)
• cheap
3’
5’
5’
3’
Extension
ID
3’
5’
Taq
ID
ID
Taq
5’
ID
ID
3’
Apply Excitation
Wavelength
l
l
3’
l
ID
ID
ID
Taq
l
ID
ID
l
Taq 5’
3’
Melting curve – Test the presence of unspecific amplification,
contamination, primer dimers,..
TaqMan probes
• Sequence-specific
• Doesn´t need much optimization
• More expensive
What Type
of
Instruments
are used
with RealTime PCR?
Real-time PCR instruments consist
of TWO main components:
• Thermal Cycler (PCR machine)
• Optical Module (to detect
fluorescence in the tubes during
the run)
What Type
of
Instruments
are used
with RealTime PCR?
• Adequate, optical plates
 96/384 wells
 Standard/fast
• Optical sealing adhesive
Quantification
and
Normalization
• First basic underlying principle: every
cycle there is a doubling of product.
Quantification and
Normalization
• Second basic principle: we do not
need to know exact quantities of DNA,
instead we will only deal with relative
quantities.
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
0
5
10
15
20
25
30
35
40
• Third basic principle: we have to have
not only a “target” gene but also a
“normalizer” gene.
• Key formula:
Quantity = 2 ^ (Cta – Ctb)
Standard Curve
Quantification and
Normalization
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
0
5
10
15
20
25
30
35
40
Prepare a 2-fold serial dilution of a DNA sample:
Recomendation: add always a standard curve in every run
“normalizer” gene
Quantification and
Normalization
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
• Knowing the amount of mRNA in one sample
from one specific gene does not tell us much..
• You need to 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 of a gene increase 2x after induction
0
0
5
10
15
20
25
30
35
40
It is possable that all (1) genexpression in the cell has
increased (2) the induced samples contained more
total mRNA
We have
to compare the expression of our
gene to another gene which expression is
normally constant, a housekeeping gene (ex.
TBP, 18S)
ΔΔCt method
experiment
control
-[(Cttg-Ctcg)-(Cttg-Ctcg)]
2
Ex!
 Ct = target gene– ref gene
 Ct = 9.70
 Ct = target gene– ref gene
 Ct = -1.70
Difference = Ct-Ct
= Ct
= 9.70-(-1.7)
= 11.40
Fold change = 211.40 = 2702
Always in duplicate or triplicate!
Quanto aos primers..
• Para qPCR: Iniciadores que flanqueiam a junção exon-exon para evitar
amplificação inespecífica devido à contaminação por gDNA.
• Sempre testar os primers pela 1ª vez por PCR normal em pelo menos 3
diferentes temperaturas e correr gel!
Escolher a máxima temperatura em que há apenas 1
banda (e do tamanho esperado!) e a amplificação ainda é
satisfatoria!
Calculando a eficiência dos primers em qPCR
Ideal: N = N0.2n
N = número de moléculas amplificadas
N0 = número de moléculas inicial
n = Número de ciclos
threshold
Ct
Curva padrão
threshold
SERIES OF 10-FOLD DILUTIONS
15
10,000,000,000
100% EFF
90% EFF
80% EFF
70% EFF
AMOUNT OF DNA
1,000,000,000
100,000,000
10,000,000
1,000,000
100,000
10,000
1,000
100
10
1
0
10
20
PCR CYCLE NUMBER
E = 10^(-1/slope)
E = (10^(-1/slope)-1)*100
Ex. slope = -3,81
Eficiência = (10^(-1/-3.81)-1)*100 = 83%
Ideal: eficiência >95%
30
CYCLE AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA
100% EFFICIENCY 90% EFFICIENCY 80% EFFICIENCY 70% EFFICIENCY
0
1
1
1
1
1
2
2
2
2
2
4
4
3
3
3
8
7
6
5
4
16
13
10
8
5
32
25
19
14
6
64
47
34
24
7
128
89
61
41
8
256
170
110
70
9
512
323
198
119
10
1,024
613
357
202
11
2,048
1,165
643
343
12
4,096
2,213
1,157
583
13
8,192
4,205
2,082
990
14
16,384
7,990
3,748
1,684
15
32,768
15,181
6,747
2,862
16
65,536
28,844
12,144
4,866
17
131,072
54,804
21,859
8,272
18
262,144
104,127
39,346
14,063
19
524,288
197,842
70,824
23,907
20
1,048,576
375,900
127,482
40,642
21
2,097,152
714,209
229,468
69,092
22
4,194,304
1,356,998
413,043
117,456
23
8,388,608
2,578,296
743,477
199,676
24
16,777,216
4,898,763
1,338,259
339,449
25
33,554,432
9,307,650
2,408,866
577,063
26
67,108,864
17,684,534
4,335,959
981,007
27
134,217,728
33,600,615
7,804,726
1,667,711
28
268,435,456
63,841,168
14,048,506
2,835,109
29
536,870,912
121,298,220
25,287,311
4,819,686
30
1,073,741,824
230,466,618
45,517,160
8,193,466
35
1
1
Results?
2.5
Good experimental design
Optimal primers
Good RNA
Good cDNA
Relative gene expression
log10 of relative gene expresion
2
1.5
Adequate nr of samples
Adequate nr of replicates
1
0.5
0
Good STATISTICS
-0.5
-1
-1.5
Treatment
Gene
A
B
C
D
E
F
G
UCE
Value
1.828745739
2.04179718
0.666738198
1.999855536
- 0.450673805
0.509327854
- 1.195371388
0
Call
up regulated
up regulated
unaffected
up regulated
unaffected
unaffected
down regulated
control gene