mRNA Quantification
Jan 23, 2016
mRNA Quantification
Real-time PCR
• To accurately quantify starting amounts of nucleic acid during the PCR reaction without the need for post-PCR analyses.
• A fluorescent reporter is used to monitor the PCR reaction as it occurs; the reporter can be of a nonspecific or specific nature.
• The fluorescence of the reporter molecule increases as products accumulate with each successive round of amplification.
RT-PCR
• Reverse transcription + Polymerase chain reaction
• mRNA quantification
Threshold Cycle (Ct)
• In the early cycles of amplification, the change in fluorescence of the reporter is usually undetectable, but at some point during amplification, the accumulation of product results in a measurable change in the fluorescence of the reaction mixture.
• The point at which the fluorescence rises appreciably above background has been called the threshold cycle.
An Example
Well CtB3 10.2B4 13.6B5 16.7B6 20.9
3.322=log210
1/10 serial dilution
Ct=log2(fold difference)
SYBR Green I
Primer 를 넣어서 annealing 한 후 , 몇몇 dye 분자들이 dsDNA 와 결합한다 .Dye 가 dsDNA 에 결합함으로 , excitation 후 극격한 형광이 발산한다 .
dsDNA 에 결합되지 않은 dye 는 아주 약한 형광을 내지만 , 컴퓨터 분석과정에서 이러한 결합되지 않은 dye 에 의한 형광값 (background)은 충분히 제거할 수 있다 .
DNA 가 증폭 되는 동안 , dye 분자가 새로이 합성되는 DNA 가닥에 결합한다 . 증가되는 형광값을 real-time 으로 볼 수 있다 .
M****
M**** expression difference between samples
Types
Reference HL-60 CCRF-CEM
Ct
18
20
22
24
26
28
30
32
34
36
10 ng mRNA template50 ng mRNA template100 ng mRNA template
10 ng 50 ng 100 ng
Reference 25.25 21.8 21.1
HL-60 23.5 21.4 19.5
CCRF-CEM 33.35 30.65
Ct difference Fold-difference
10 ng 50 ng 100 ng 10 ng 50 ng 100 ng
HL-60 -1.75 0.4 -1.6 3.36 1.32 3.03
CCRF-CEM 8.1 8.85 274.37 461.44
일단 , Ct 값이 좀 커서 .. Unreliable 하다
Gene Symbol
HL60CCRF-CEM
M differenc
e
Fold-differenc
e
M****1.53574
5-1.68172
3.217465
9.301511
A**** -1.96497 -0.09118 -1.873790.27285
6
C****1.11582
2-1.17847
2.294288
4.905118
D**** -0.136670.06064
1-0.19731
0.872175
M**** expression difference between samples
Type
Reference HL-60 CCRF-CEM
Ct (
Th
resh
old
cycl
e)
20
22
24
26
28
30
32
34
36
CtCt
difference
fold differenc
e
Reference
23.8 0 0
HL-60 22.5 -1.32.46228
9
CCRF-CEM
34.6 10.80.00056
1
이것도 , Ct 값이 좀 커서 .. Unreliable
하다
2nd experiment
Gene Symbol
HL60CCRF-CEM
M differenc
e
Fold-differenc
e
M****1.53574
5-1.68172
3.217465
9.301511
A**** -1.96497 -0.09118 -1.873790.27285
6
C****1.11582
2-1.17847
2.294288
4.905118
D**** -0.136670.06064
1-0.19731
0.872175
Effect of mRNA concentration on RT
mRNA concentration (ng/l)
0 20 40 60 80 100
Ct (T
hre
shol
d c
ycle
)
18
20
22
24
26
28
30
32
34
36
Reference sampleHL-60CCRF-CEM
25.2
21.8 21.1
33.35
30.65
23.5
21.4
19.5
2.722.7=6.50
3.423.4=10.56
0.720.7=1.62
2.122.1=4.29
1.921.9=3.73
Effect of mRNA concentration on RT (fold-difference; MGST1)
mRNA concentration (ng/l)
0 20 40 60 80 100
Fol
d-d
iffe
ren
ce
0
2
4
6
8
10
12
14
16
18
ReferenceHL-60CCRF-CEM
mRNA concentration effect (x-axis in logrithm)
log (mRNA concentration)
0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
Ct (
Th
resh
old
cyc
le)
18
20
22
24
26
28
30
32
34
36
ReferenceHL-60CCRF-CEM
Effect of mRNA concentration on RT
log (mRNA concentration (ng/l))
0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
Ct (T
hre
shol
d c
ycle
)
16
18
20
22
24
26
28
30
ReferenceHL-60x column vs y column
기울기가 .. 좀 크다 . - 4 정도
2nd experiment
A****
A**** expression level difference between samples
Types
Reference HL-60 CCRF-CEM
Ct (
Thr
esh
old
cyc
le)
26.5
27.0
27.5
28.0
28.5
29.0
29.5
30.0
30.5
Col 7 vs Col 10
27.5
30.3
26.8
2.822.8=6.96
0.720.7=1.62
이것도 Ct 값이 좀 커서 .. Unreliable 하다
차이가 좀 크긴 하네 .. 거의 같게 나와야
하는데
Gene Symbol
HL60CCRF-CEM
M differenc
e
Fold-differenc
e
M****1.53574
5-1.68172
3.217465
9.301511
A**** -1.96497 -0.09118 -1.873790.27285
6
C****1.11582
2-1.17847
2.294288
4.905118
D**** -0.136670.06064
1-0.19731
0.872175
Effect of mRNA concentration on RT
mRNA concentration (ng/l)
0 20 40 60 80 100 120
Ct (
Th
resh
old
cyc
le)
24
25
26
27
28
29
30
31
Reference sampleHL-60CCRF-CEM
27.5
25.85
25.2
1.6521.65=3.14
0.6520.65=1.57
1.821.8=3.48
0.520.5=1.41
30.3
28.5
28
Effect of mRNA concentration on RT (A****)
log (mRNA amount)
0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
Ct (
Th
resh
old
cyc
le)
24
25
26
27
28
29
30
31
ReferenceHL-60
x column 1 vs y column 1
Rsqr = 0.992y = 32.61-2.348x
Rsqr = 0.9996y = 29.80-2.311x
Slope of 2.33 means the …
농도에 따른 경향은 일정한데 , 그 slope 이 너무 작다 .
이론적으로는 3.322 가 나와야 한다 .
Effect of mRNA concentration on RT (fold-difference; A****)
mRNA concentration (ng/l)
0 20 40 60 80 100
Fol
d-d
iffe
ren
ce
0
1
2
3
4
5
6
ReferenceHL-60
mRNAReferenc
eHL-60
100 4.9246 4.9246
50 3.1383 3.4822
10 1 1
C****
C**** expression level difference between samples
Types
Reference HL-60 CCRF-CEM
Ct (
Th
resh
old
cycl
e)
26
27
28
29
30
31
27.0
26.45
30.45
0.5520.55=1.464
424=16
Gene Symbol
HL60CCRF-CEM
M differenc
e
Fold-differenc
e
M****1.53574
5-1.68172
3.217465
9.301511
A**** -1.96497 -0.09118 -1.873790.27285
6
C****1.11582
2-1.17847
2.294288
4.905118
D**** -0.136670.06064
1-0.19731
0.872175
C**** expression level difference between samples (10 ng/l sample)
Type
Reference HL-60 CCRF-CEM
Ct (T
hre
shol
d cy
cle)
28
29
30
31
32
33
Gene Symbol
HL60CCRF-CEM
M differenc
e
Fold-differenc
e
M****1.53574
5-1.68172
3.217465
9.301511
A**** -1.96497 -0.09118 -1.873790.27285
6
C****1.11582
2-1.17847
2.294288
4.905118
D**** -0.136670.06064
1-0.19731
0.872175
2nd experiment
C**** expression level difference between reference and HL-60
Types
Reference HL-60
Ct (
Th
resh
old
cyc
le)
23
24
25
26
27
28
100 ng/l50 ng/l10 ng/l
100 ng/ml
50 ng/ml 10 ng/ml
Reference
25.45 25.05 26.45
HL-60 24.25 24.1 27
Ct differenc
e1.1 0.95 -0.55
Fold-differenc
e2.14 1.93 0.68Gene
SymbolHL60
CCRF-CEM
M differenc
e
Fold-differenc
e
M****1.53574
5-1.68172
3.217465
9.301511
A**** -1.96497 -0.09118 -1.873790.27285
6
C****1.11582
2-1.17847
2.294288
4.905118
D**** -0.136670.06064
1-0.19731
0.872175
Effect of mRNA concentration on RT
mRNA concentration (ng/l)
0 20 40 60 80 100 120
Ct (
Th
resh
old
cyc
le)
22
24
26
28
30
32
Reference sampleHL-60CCRF-CEM
Effect of mRNA concentration on RT
mRNA concentration (ng/l)
10 100
Ct (
Th
resh
old
cyc
le)
22
24
26
28
30
32
Reference sampleHL-60CCRF-CEM
Effect of mRNA concentration on RT (fold-difference; C****)
mRNA concentration (ng/l)
0 20 40 60 80 100
Fol
d-d
iffe
ren
ce
0
1
2
3
4
5
6
7
8
ReferenceHL-60
mRNAReferenc
eHL-60
100 2.0000 6.2767
50 2.6390 7.4643
10 1 1
Effect of mRNA concentration on RT
mRNA concentration (ng/l)
0 20 40 60 80 100
Ct (T
hres
hold
cyc
le)
24
26
28
30
32
34
Col 8 vs Col 9 Col 8 vs Col 11 Col 8 vs Col 12
mRNA conc.
log (mRNA
)Reference HL-60
CCRF-CEM
CtStd. Dev.
Ct Ct
100 2 26.3 0.2828 25.1
50 1.699 26.55 0.6364 25.8
10 1 28.65 0.0707 28.8 32.4
2nd experiment
2D Graph 3
log (mRNA concentration)
0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
Ct (T
hres
hold
cyc
le)
24
25
26
27
28
29
Col 7 vs Col 9 Col 7 vs Col 11 x column vs y column
2nd experiment
D****
D**** expression level difference between samples
Type
Reference HL-60 CCRF-CEM
Ct (T
hre
shol
d cy
cle
)
24.5
25.0
25.5
26.0
26.5
27.0
25.75
24.85
26.45
0.920.9=1.87
0.720.7=1.62
Gene Symbol
HL60CCRF-CEM
M differenc
e
Fold-differenc
e
M****1.53574
5-1.68172
3.217465
9.301511
A**** -1.96497 -0.09118 -1.873790.27285
6
C****1.11582
2-1.17847
2.294288
4.905118
D**** -0.136670.06064
1-0.19731
0.872175
D**** expression level difference between reference and HL-60
Types
Reference HL-60
Ct (
Th
resh
old
cyc
le)
23.0
23.5
24.0
24.5
25.0
25.5
26.0
26.5
27.0
100 ng/l50 ng/l10 ng/l
100 ng/ml 50 ng/ml 10 ng/ml
Reference
24.65 23.95 25.75
HL-60 23.6 23.5 26.45
Ct differenc
e1.05 0.45 -0.7
Fold-differenc
e2.07 1.37 0.616Gene
SymbolHL60
CCRF-CEM
M differenc
e
Fold-differenc
e
M****1.53574
5-1.68172
3.217465
9.301511
A**** -1.96497 -0.09118 -1.873790.27285
6
C****1.11582
2-1.17847
2.294288
4.905118
D**** -0.136670.06064
1-0.19731
0.872175
Effect of mRNA concentration on RT
mRNA concentration (ng/l)
0 20 40 60 80 100 120
Ct (
Thr
esh
old
cycl
e)
23.0
23.5
24.0
24.5
25.0
25.5
26.0
26.5
27.0
Reference sampleHL-60CCRF-CEM
25.75
23.95
23.6
1.821.65=3.48
- 0.12-0.1=0.933
2.9522.95=7.73
- 0.52-0.5=0.62
26.45
23.5
24.65
Effect of mRNA concentration on RT (D****)
log (mRNA amount)
0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
Ct (
Thr
esho
ld c
ycle
)
23.0
23.5
24.0
24.5
25.0
25.5
26.0
26.5
27.0
ReferenceHL-60
Effect of mRNA concentration on RT (fold-difference; D****)
mRNA concentration (ng/l)
0 20 40 60 80 100
Fol
d-di
ffere
nce
0
2
4
6
8
10
ReferenceHL-60
mRNAReferenc
eHL-60
100 2.1435 7.2100
50 3.4822 7.7275
10 1 1
Discussion
• Repetitive experiments• mRNA concentration dependency• t-values, are they still valid?