Analysis of data from Real

time experiments

J.M.K. Mulema

Department of Molecular and Cell Biology

University of Cape Town

• Quantifying RNA; Northern blotting, In situ hybridization, RNAse

protection assays, Microarray, RT-PCR.

• Real time PCR

– Data collected throughout the PCR process as it occurs.

– Amplification and detection combined into a single step.

– Reactions characterized by the point in time where the target amplification

is first detected.

– Cycle threshold (Ct), the time at which fluorescent intensity is greater than

background fluorescence.

– Greater starting target DNA, faster significant increase in fluorescent,

lower Ct

– Requires much less RNA template

Introduction

One step Vs Two step

One step

• cDNA synthesis to PCR amplification is

performed in a single tube.

• Minimizes experimental variation

because both enzymatic reactions

occur in a single tube

• Uses RNA starting template. Prone to

rapid degradation if not handled well.

• Not suitable in situations where the

same sample is assayed on several

occasions over a period of time.

• Less sensitive than two step protocols.

Two step

• Reverse transcription and PCR

occur in separate tubes

• Allows several different real time

PCR assays on dilutions of a single

cDNA.

• Reactions from subsequent assays

have the same amount of template

to those assayed earlier.

• Date from two step is quite

reproducible.

• Allow for increased DNA

contamination. Design the target

PCR product to span introns

• Genome sequenced

• A rapid life cycle

• Prolific seed production

• Cultivation in restricted space

• Easily transformed

• Botrytis cinerea (anamorph)

• Botryotinia fuckeliana (teleomorph)

• Ubiquitous

• Chemical control

• Host range

Identify genes that play a

role in resistance

• Arabidopsis leaves infected with B. cinerea

• Extracted RNA from first three replicates

(untreated, 12 hrs pi and 24 hrs pi)

• Made overnight cDNA synthesis

• Used Superscript III reverse transcriptase in a

20µl reaction.

• Diluted the synthesized cDNA 1 in 10

• Designed primers to amplify products ranging

from 70-150 bp

• Amplification optimized with a conventional

PCR before real time.

1. At3g51660 (Macrophage migratory)

2. At4g30270 (MERI-5 protein)

3. At2g47190 (Myb family transcription)

4. At5g39610 (No Apical Meristem)

5. At5g06860 (PGIP1)

6. At4g24340 (Phosphorylase)

7. At5g07010 (Sulfotransferase)

8. At1g22400 (UDP-glucoronosyl)

9. At1g62300 (WRKY)

10. At3g04720 (Hevein-like protein)

11. At3g28930 (avrRpt2-induced protein)

12. At3g50480 (Broad spectrum)

13. At2g24180 (CYP 450)

14. At3g04220 (Disease resistance protein)

15. At1g52200 (Expressed protein)

16. At2g39030 (GCN5-related acetyltransferase)

17. At4g16260 (Glycosyl hydrolase)

18. At4g15610 (Integral membrane protein)

19. At4g33150 (lysine-ketoglutarate)

1. At4g10340 (Chlorophyll A-B)

2. At1g72610 (Germin-like protein)

3. At1g12900 (GAPHD)

4. At5g38430 (RUBSCO)

5. At1g20340 (Plastocyanin)

1. At5g25760 (Ubiquitin-conjugating enzyme)

2. At5g06600 (Ubiquitin-specific protease)

3. At1g04820 (Tubulin alpha 2-alpha 4)

4. At5g44200 (Nuclear cap-binding protein)

• Absolute/Relative quantification

• Used serial diluted standards of

known concentration to generate a

standard curve.

• Standard curve is a linear

relationship between the ct and the

initial amount amounts of RNA or

cDNA.

• This allows the determination of the

concentration of unknowns based on

their ct values

• Assumes that all standards and

samples have equal amplification

efficiencies.

• The concentrations of serial dilutions

should encompass all samples and

stay within the range that can be

quantified

Rep 10 hrs12 hrs pi24 hrs pi

Rep 20 hrs12 hrs pi24 hrs pi

Rep 30 hrs12 hrs pi24 hrs pi

Pooled sample

Dilution 1 (10-1)

Dilution 2 (10-2)

Dilution 3 (10-3)

Generating a standard curve

Carry out runs in triplicates

Source: Celtic Diagnostic

Gene 0hrs R1 12hrs R1 24hrs R1 0hrs R2 12hrs R2 24hrs R2 0hrs R3 12hrs R3 24hrs R3

At3g28930 11.756 81.5393 224.747 5.1133 33.1119 388.9217 8.6277 85.026 106.6237

At3g50480 64.3495 158.3122 179.2381 54.568 70.2542 186.2909 48.3419 101.5222 109.3803

At2g24180 13.7458 55.4771 309.4145 13.5755 33.9872 292.9062 14.5349 77.8196 141.6806

At3g04220 29.3513 93.8687 359.3015 12.8654 29.3513 355.5778 12.2984 168.3563 156.9873

At1g52200 18.0219 111.6761 158.4963 15.463 56.2404 242.5491 14.948 210.0839 168.0951

At2g39030 0.3443 59.346 305.0308 0.3303 32.7631 644.4388 0.2339 58.1566 447.241

At4g16260 0.2982 13.2753 365.5219 0.0909 3.1700 330.4173 0.0922 12.0322 363.5908

At3g04720 4.161 22.9246 372.9041 0.9894 10.1421 316.1588 1.2689 22.79 361.511

At4g15610 2.4848 42.4467 395.1967 2.703 27.3786 302.0836 2.8432 57.5879 260.4722

At4g33150 2.6385 48.3277 269.8711 5.0124 105.9577 137.7378 4.0888 45.0527 294.8066

At3g51660 7.688 71.5792 328.3608 7.9407 56.4595 214.9203 6.4393 109.8782 247.967

At4g30270 12.3045 99.6367 197.8529 22.8425 65.073 185.0064 16.0316 175.1437 319.8013

At2g47190 2.6065 43.7507 325.6823 2.1613 43.8846 315.7555 1.9861 52.0448 333.4451

At5g39610 3.2338 135.7344 293.7087 10.2306 123.9325 909.3652 2.451 212.3385 463.2975

At5g06860 10.4241 62.6886 171.8571 31.3713 39.1112 355.6503 27.1353 112.4979 310.3416

At4g24340 21.4914 27.9686 277.4395 9.145 19.2327 436.6081 13.63 52.4527 301.7165

At5g07010 0.2782 78.6687 261.2367 0.5103 78.1067 250.2366 0.3266 92.4533 203.6362

At1g62300 19.4567 58.0412 334.387 13.6397 42.2785 530.477 17.7916 156.3018 174.7055

At1g22400 17.3066 40.4971 454.7851 11.0579 12.2543 632.0124 10.099 115.3882 250.8585

Upregulated genes

Gene 0hrs R1 12hrs R1 24hrs R1 0hrs R2 12hrs R2 24hrs R2 0hrs R3 12hrs R3 24hrs R3

At1g20340 309.6129 73.8873 9.5136 237.8231 67.4855 9.9133 307.8662 43.0424 30.1606

At1g72610 399.3349 52.4858 0.3119 255.9348 29.857 0.8784 360.056 50.771 3.6052

At1g12900 484.7891 32.245 1.8623 236.421 22.1902 3.5271 256.4948 11.8885 10.5508

At5g38430 419.7972 26.3733 0.4 168.4723 16.1544 1.9259 221.1612 7.4169 1.8047

At4g10340 204.9719 73.5833 2.8063 266.3565 84.4429 7.0391 194.7971 80.9538 18.1863

Down regulated genes

Gene 0hrs R1 12hrs R1 24hrs R1 0hrs R2 12hrs R2 24hrs R2 0hrs R3 12hrs R3 24hrs R3

At5g44200 121.2694 103.3107 67.5263 123.0522 107.1146 65.1304 121.7226 112.7576 102.7511

At5g25760 127.6356 183.0081 118.7417 160.7317 195.4594 184.7387 166.7819 156.2693 154.9688

At1g04820 157.1404 180.7631 34.8381 166.0978 102.6774 112.9576 189.3141 153.4388 34.0457

At5g06600 64.3015 104.2577 94.2011 69.823 85.3205 177.1222 53.1897 61.0233 74.8282

Reference genes (Relative Conc)

Gene 0hrs R1 12hrs R1 24hrs R1 0hrs R2 12hrs R2 24hrs R2 0hrs R3 12hrs R3 24hrs R3

At5g44200 20.16 20.4 21.05 20.14 20.35 21.1 20.16 20.27 20.41

At5g25760 20.83 20.38 20.92 20.55 20.3 20.37 20.5 20.58 20.59

At1g04820 15.57 15.37 17.72 15.49 16.18 16.04 15.3 15.6 17.76

At5g06600 18.19 17.38 17.55 18.05 17.71 16.5 18.5 18.27 17.93

Reference genes (Ct values)

• Absolute quantification

– Uses serially diluted standards of known concentration to

generate a standard curve.

– PCR standards

• Fragment of double stranded DNA

• Single stranded DNA

• Complementary RNA bearing the target sequence

• Relative quantification

– Changes in gene expression are an external standard or

calibrator

• Two standard curve method

• Comparative Ct (Delta Delta Ct) method

• Pfaffl method – Relative expression software tool (REST)

Types of real-time quantification

Replicate GOI HKG GOI/HKG Relative values

0 hrs Rep 1 13.7458 127.6356 0.107695659 1.000

12 hrs Rep 1 55.4771 183.0081 0.303140134 2.815

24 hrs Rep 1 309.4145 118.7417 2.60577792 24.196

0 hrs Rep 2 13.5755 160.7317 0.084460626 1.000

12 hrs Rep 2 33.9872 195.4594 0.173883681 2.059

24 hrs Rep 2 292.9062 184.7387 1.585516191 18.772

0 hrs Rep 3 14.5349 166.7819 0.087149145 1.000

12 hrs Rep 3 77.8196 156.2693 0.497983929 5.714

24 hrs Rep 3 141.6806 154.9688 0.914252417 10.491

Calculation of relative values (At2g24180)

0

50

100

150

200

250

300

350

0 hrs Rep 1 12 hrs Rep 1 24 hrs Rep 1 0 hrs Rep 2 12 hrs Rep 2 24 hrs Rep 2 0 hrs Rep 3 12 hrs Rep 3 24 hrs Rep 3

Time points

Rel

ativ

e va

lues

At4g15610 At4g33150 At2g47190 At5g39610 At3g04720

0

0.2

0.4

0.6

0.8

1

1.2

0 hrs Rep 1 12 hrs Rep 1 24 hrs Rep 1 0 hrs Rep 2 12 hrs Rep 2 24 hrs Rep 2 0 hrs Rep 3 12 hrs Rep 3 24 hrs Rep 3

Time points

Rel

ativ

e va

lues

At4g10340 At1g72610 At1g12900 At5g38430 At1g20340

Relative expression software tool (REST)

• Purpose is to determine if there is a significant difference between samples and

controls taking into account issues of reaction efficiency and reference gene

normalization.

• Randomization are used in Rest.

• The alternate hypothesis P(H1) is based on the assumption that the difference

between sample and control groups is due only to chance.

• REST performs 50,000 random reallocations of samples and controls between the

groups and counts the number of times the relative expression of the randomly

assigned group is greater than the sample data

• Concentration = efficiencyavg(Controls) – avg(Samples)

• Expression = goiConcentration ÷ refConcentration

• Expression = GEOMEAN(goiConcentration ÷ refConc1, goiConcentration ÷

refConc2, …)

• Calculate a normalization factor equal to the geometric mean

• REST performs its calculations based on CP and efficiency values determined by

standard curve or kinetic techniques.

Validated genes from Microarray

1.09

E-0

1

2.66

E+0

1

9.04

E+0

1

3.64

E-0

1

2.27

E+0

0

3.72

E+0

0

7.68

E-0

3

5.79

E-0

1

3.49

E+0

1

1.88

E+0

2

6.53

E+0

0

4.24

E-0

1

0.00E+00

2.00E+01

4.00E+01

6.00E+01

8.00E+01

1.00E+02

1.20E+02

1.40E+02

1.60E+02

1.80E+02

2.00E+02

0 hrs 12 hrs 24 hrs 0 hrs 12 hrs 24 hrs 0 hrs 12 hrs 24 hrs 0 hrs 12 hrs 24 hrs

Mea

n N

orm

aliz

ed E

xpre

ssio

n

Delta Delta Ct method

• Xn = Xo x (1 + Ex)n

• XT = Xo x (1 + Ex)CT,X =KX

• RT = Ro x (1 + ER)CT,R =KR

Dividing XT by RT gives the expression

XT = Xo x (1 + Ex)CT,X =KX

RT = Ro x (1 + ER)CT,R =KR

Assuming equal amplification efficiencies

EX =ER = E

Xo

Ro

x (1 + E) CT,X-CT,R

= XN x (1 + E) ΔCT

= XNcb x (1 + E) ΔCT,cb

= XN,q x (1 + E) ΔCT,q = (1 + E) -

ΔΔCT

Calibration

Amount of the target normalized to an endogenous reference and relative to the calibrator is given by

Amount of target = 2-ΔΔCT

Assumption: The amplification efficiency of the target and reference are approximately equal

y = -0.175x - 2.8609

R2 = 0.3827

-4

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

0

0 0.5 1 1.5 2 2.5

Log cDNA Conc

CT

(T

arg

et -

Ref

)

Time GAPDH UBC ΔCT -ΔΔCT Amount of target

0 hrs 15.16 20.83 -5.67 1

0 hrs 16.28 20.55 -4.27 1

0 hrs 16.15 20.5 -4.35 1

12 hrs 19.38 20.38 -1 4.67 0.04

12 hrs 19.96 20.3 -0.34 3.93 0.07

12 hrs 20.93 20.58 0.35 4.7 0.04

24 hrs 23.82 20.92 2.9 8.57 0.003

24 hrs 22.82 20.37 2.45 6.72 0.009

24 hrs 21.12 20.59 0.53 4.88 0.033

0

0.2

0.4

0.6

0.8

1

1.2

0 hrs 0 hrs 0 hrs 12 hrs 12 hrs 12 hrs 24 hrs 24 hrs 24 hrs

Amount of Target

Tim

e poin

ts

• Real Time PCR is a powerful technique that

gives quantitative answers difficult to

obtain with end point PCR, however, all

steps need to be controlled from sampling

to PCR including manipulations like

extraction and reverse transcription

Conclusion