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?