Tutorial: Transcriptomics Case Study
EFFECT OF CALORIE
RESTRICTION ON GENE
EXPRESSION PROFILE IN HUMAN
ADIPOSE TISSUE
Nathalie Viguerie, 2007 June 8th
Transcriptomics Case Study
Contents
slides
Designing the experiment
PCA analyses of clinical data
Microarrays: preparations and data outputs
Data normalization and PCA analysis of gene data
Genes over- and under-expressed
Ontological analysis
Summary and conclusions
Abbreviations used
3-6
7-13
11-22
23-30
31-38
39-43
44-48
49
Project Diogenes
RTD 2: Genetics/Genomics
Obesity and Gene-Nutrient Interaction
•Identification of gene-nutrient interactions associated with changes in
body weight
•Characterization of biomarkers of nutritional status and predictors of
weight and metabolic variation
•Genetic, transcriptomic, peptidomic and proteomic
First step
Identification of potential mRNA predictors of weight change (INSERM
Paris) and mRNA biomarkers of nutritional status (INSERM Toulouse,
Lyon) with the RNA biobank from the NUGENOB cohort (750 subjects).
10 weeks of a low calorie diet (- 600 kcal /day).
Low fat/high carbohydrate diet and High fat/low carbohydrate diet
Transcriptomic approach to
compare gene expression
before and after LCD
Pangenomic cDNA
arrays
Experimental design
Low fat/ High CHO diet
High fat / Low CHO diet
fat 40
fat 20
- 45%
- 25%
carbohydrate
60 - 65%
protein 15%
carbohydrate
40 - 45%
protein 15%
2 x 24 obese subjects
2 x 24 obese subjects
1 set for microarray experiments
1 set for RT-PCR confirmatory analyses and
determination of potential candidate genes
Subject selection
Selection criteria:
Completed weight loss intervention
Female
Caucasian
Pre-menopausal
No medication
No diabetes
Genotyping data available
Good quality RNA at baseline and week 10
Food recording data available at baseline and during intervention
To create 2 groups as closely matched as possible for analysis of effect of
different diet composition, we have selected the following:
• Fat Energy % during intervention : 18-28% for LF and 37-48 % for MF.
• Weight loss between 4 and 10 kg (exclude extremes)
• Have an initial self-reported energy intake of more than 4000 kcal/day.
• Reported reduction in energy intake was less than 1300 kcal/day.
Following this procedure we have succeeded in selecting 55 subjects from the
‘low fat’ group and 49 subjects from the ‘high fat’ group.
RTD
Principal Components Analysis
(PCA ) : clinical data
48 subjects (24LF - 24HF) and 20 clinical data (log ratio) :
1
2
3
4
5
6
7
8
9
10
weight
BMI
Fat.Free.Mass
Fat.Mass
Fat.mass %
free.fatty.acids
glucose
tot.cholesterol
HDL.cholesterol
VLDL.cholesterol
11
12
13
14
15
16
17
18
19
20
LDL.cholesterol
Tot.glycerol
Free.glycerol
Triglycerides
Insulin
Leptin
Cortisol
Insulin.like.growth.factor.1
HOMA.Insulin.secretion
HOMA.Insulin.resistance
www.diogenes-eu.org
PCA : clinical data (SIMCA-P®)
Plot of the first ten eigenvalues of the PCA
Axis
1
2
3
4
5
6
7
8
9
10
Proportion of
variance
0.361
0.21
0.112
0.097
0.085
0.049
0.029
0.023
0.016
0.011
Cumulative
proportion
0.361
0.57
0.681
0.778
0.863
0.912
0.941
0.964
0.980
0.991
68% information with 3 components
www.diogenes-eu.org
RTD
PCA : clinical data (SIMCA-P®)
www.diogenes-eu.org
RTD
PCA : clinical data (SIMCA-P®)
www.diogenes-eu.org
Total RNA quality check
Adipose tissue biopsies obtained before and after the LCD
Go/NoGo: quality checked by Agilent Bioanalyzer
IVT
Go
Amplification + indirect labeling
with Cy3 or Cy5
Combining equal
amounts
Control of dye incorporation
cDNA microarray
Control of dye incorporation
Cy3 labelled sample: Nanodrop ND1000’s dye
absorbance quantification
Cy3
detection
Nucleic acid
detection
Cy5
detection
cDNA yield and
specific activity
calculation
allows detection at dye concentration as low as 0.2 pmol/ul
Dye incorporation calculation
• Other requirement: comparable incorporation for each dye in the
respective cDNA samples
CyScribe post-labelling Kit
(Amersham Bioscience)
Yield pmol Cy per
µg cDNA
FOI (frequence of
incorporation)
Recommendation
80-200
20-70
Yield: pmol dye /µg nucleic acid
FOI: (pmol dye x 324.5)/ng cDNA
• 2 out of 48 labelings needed new experiments because of a low
cyanine incorporation and a strong imbalance Red-Green
• 4 out of 48 experiments showed a low valid spot% (detection after
hybridization) and were remade
Microarray experiments
Combining equal amounts of each dye +
cDNA microarray
washes
Detection of Cy-3 and Cy-5
emission signals
(fluorescence)
scanning
TIFF
image
Map the location of the pixels of each spot
Calculation of the intensity values of each probe and the quantitative ratio of the
fluorescence intensity levels between the two samples. Genepix software
cDNA vs oligonucleotide chip
hybridization images
cDNA array
Oligonucleotide array
Poor quality hybridization
This experiment needed a new hybridization
Image quantification and data
filtering
Genepix software
Grid file placement
(feature identification)
Signal saturation
Feature finding,adjustment and flagging
flagged
Not found
Saturated: flagged
Output of the data (GPR file)
Open with Excel: 41761 rows and 56 columns !!
< 10 columns used for data filtering and normalization
What is important for feature signal intensity ?
D,E
Gene
Identification
2 columns
J
O
Red signal
feature
background
2 similar columns for the green signal (columns V
and AA)
Flagged spots identification
(irregular or saturated features)
BB
These features were flagged during image quantification.
Won’t be used when expression ratio is retrieved
(-50 = not found;-75= absent; -100=bad spot; +100= flag good)
DATA NORMALIZATION
Lowess Normalization effect
Print-tip consideration
Normalization
• Different procedures:
– housekeeping genes
– spiking
– total intensity normalization
– complex normalization
• lowess fitting with or without stratification (different sector of the arrays
should be separately normalized). Adjustment of the center of log
ratios.
MA plot (Log2(R/G)=f(Log10(RxG))
Lowess normalization
M = Log2(R/G)
A = Log10(RxG)
Box-plots advantages
2 microarrays are outliers
median
Box plot of R/G ratios by array
After Lowess
Log2 ratio
Before Lowess
Normalization: allows data centering
RTD
PCA : gene data
48 subjects (24LF - 24HF) and 19554 genes after normalization.
Plot of the first ten eigenvalues of the PCA
Axis
1
2
3
4
5
6
7
8
9
10
Proportion of
variance
0.235
0.092
0.060
0.052
0.038
0.029
0.028
0.024
0.022
0.021
Cumulative
proportion
0.235
0.33
0.387
0.439
0.477
0.506
0.534
0.558
0.58
0.601
33% of the information explained with 2 components
www.diogenes-eu.org
RTD
PCA : gene data (R)
with the first 5 genes contributing more for the first two axes
www.diogenes-eu.org
RTD
PCA : gene data (SIMCA-P®)
www.diogenes-eu.org
Some exploratory analyses
Very few differential genes relating to the
fat/carbohydrate ratio.
Calorie restriction more important than
macronutrient content
Gene expression changes:
effect of calorie restriction
•Statistical analysis of microarray
•Considering unflagged spots, features with
asignal to noise ratio>2.5
RTD
delta
# med false pos
SAM : delta table
Delta Table
median
#
called
FDR
90th perc false
pos
90th perc FDR
cutlo
delta
# med false pos
90th perc false
pos
#
called
median
FDR
90th perc FDR
cutlo
cuthi
0,952661
0,916373
0,880789
0,84591
0,811735
0,778265
0,7455
0,713439
0,682083
0,651432
0,621485
0,592242
0,563705
0,535872
0,508744
0,48232
0,456601
0,431587
0,407277
0,383672
0,360771
0,338575
0,317084
0,296297
0,276215
0,256838
0,238165
0,220197
0,202934
0,186375
0,170521
0,155371
0,140926
0,127186
0,11415
0,101819
0,090193
0,079271
0,069054
0,059541
0,050733
0,04263
0,035232
0,028538
0,022548
0,017263
0,012683
0,008808
0,005637
0,003171
0,001409
0,000352
0
5,133322938
5,988876761
7,699984407
9,411092053
12,40553043
16,68329955
20,96106866
26,94994542
36,36103748
46,19990644
60,31654452
74,4331826
88,54982068
116,3553199
139,0274962
186,9385103
226,2939862
274,6327772
335,8048755
390,9880971
464,5657259
571,0821768
666,904205
770,4262176
900,4703987
1024,95348
1167,830968
1383,858309
1629,830033
1961,784916
2407,956235
2957,649566
3959,503093
4666,19055
5444,316752
6490,231301
7437,75716
8482,816155
9805,930142
10902,75014
11745,47066
12017,53677
12221,15858
12388,41936
12486,80805
12660,48547
12813,62961
12961,21264
13093,39571
13146,01227
13205,47326
13244,82873
13251,24539
32,08326836
45,60101876
57,40766152
74,51873798
97,01980352
118,151983
139,198607
166,9185509
201,3118145
250,8483809
312,5338115
371,9092468
440,6957742
511,5356308
602,3098914
737,5729508
864,3660273
982,2613441
1143,019907
1277,512968
1462,483705
1732,15427
1967,08935
2166,946723
2419,249545
2645,030199
2936,004054
3296,534435
3693,425854
4243,803628
4890,003431
5519,691044
6528,474557
7188,619887
7953,313894
8878,338687
9684,355944
10570,7097
11592,32652
12419,98929
13008,2681
13233,10765
13374,27403
13512,27486
13592,52581
13727,5322
13852,44306
13965,80394
14058,1182
14089,00369
14135,03249
14160,27132
14163,18021
541
599
636
677
708
743
774
818
860
930
1008
1070
1136
1237
1323
1472
1595
1693
1842
1948
2089
2351
2531
2683
2890
3065
3275
3594
3958
4435
5019
5708
6834
7568
8429
9599
10515
11604
12939
14023
14858
15091
15260
15404
15480
15628
15774
15915
16041
16075
16126
16155
16160
0,009488582
0,009998125
0,012106894
0,01390117
0,017521936
0,02245397
0,027081484
0,032946144
0,042280276
0,049677319
0,059837842
0,069563722
0,077948786
0,094062506
0,105085031
0,126996271
0,141877107
0,162216643
0,182304493
0,200712576
0,222386657
0,242910326
0,263494352
0,287151032
0,311581453
0,334405703
0,356589609
0,38504683
0,411781211
0,442341582
0,479768128
0,518158649
0,579382952
0,616568519
0,645903043
0,676136191
0,707347329
0,731025177
0,757858424
0,777490561
0,790514918
0,796338001
0,800862293
0,804233923
0,806641347
0,810115528
0,812325955
0,814402302
0,816245602
0,817792365
0,818893294
0,819859408
0,820002809
0,05930364
0,07612858
0,09026362
0,11007199
0,13703362
0,15902017
0,17984316
0,20405691
0,23408351
0,26972944
0,31005338
0,34757874
0,38793642
0,41352921
0,45526069
0,50106858
0,54192227
0,58018981
0,62053198
0,65580748
0,70008794
0,7367734
0,77719848
0,80765812
0,83711057
0,86297886
0,89648979
0,91723273
0,93315459
0,95688921
0,97429835
0,96700964
0,95529332
0,94987049
0,94356553
0,92492329
0,92100389
0,91095396
0,89592136
0,88568704
0,875506
0,87688739
0,87642687
0,8771926
0,87807014
0,87839341
0,87818201
0,8775246
0,87638665
0,87645435
0,8765368
0,87652562
0,87643442
-2,351
-2,284
-2,231
-2,179
-2,132
-2,084
-2,039
-1,99
-1,943
-1,891
-1,836
-1,786
-1,743
-1,693
-1,646
-1,593
-1,544
-1,502
-1,453
-1,413
-1,368
-1,317
-1,275
-1,236
-1,188
-1,147
-1,108
-1,065
-1,026
-0,979
-0,926
-0,881
-0,825
-0,783
-0,744
-0,708
-0,666
-0,631
-0,602
-0,57
-0,543
-0,508
-0,48
-0,457
-0,442
-0,419
-0,397
-0,377
-0,359
-0,353
-0,345
-0,341
-0,34
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
3,5500644
3,1950525
3,1950525
3,1950525
2,7635623
2,5868176
2,5054882
2,3087747
2,2369046
2,1696993
2,089767
2,0287648
1,9607458
1,8401803
1,772953
1,7172366
1,6695726
1,6252992
1,5628222
1,481335
1,3879799
1,2931184
1,1975641
1,085506
0,9311183
0,8379125
0,7312739
0,5929879
0,4991773
0,3846652
0,2438373
0,1358228
0,0522468
0,0503664
0,0503664
0,0503664
0,0503664
0,0503664
0,0503664
0,0503664
0,0503664
0,0503664
0,0503664
0,0503664
0,0503664
cuthi
3,523156
3,453045
3,383639
3,314937
3,24694
3,179648
3,11306
3,047177
2,981999
2,917525
2,853756
2,790692
2,728332
2,666677
2,605726
2,54548
2,485939
2,427102
2,36897
2,311543
2,25482
2,198802
2,143488
2,088879
2,034975
1,981775
1,92928
1,87749
1,826404
1,776023
1,726346
1,677374
1,629107
1,581545
1,534687
1,488533
1,443085
1,398341
1,354301
1,310966
1,268336
1,226411
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0,855553823
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0,855553823
0,855553823
0,855553823
1,711107646
2,566661469
3,507770674
0
2
3
5
5
6
6
8
10
11
12
12
14
14
16
17
19
23
26
29
29
39
46
47
56
62
69
78
79
89
105
109
117
145
150
163
196
217
227
245
264
287
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0,002981024
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0,00436507
0,00394264
0,00376896
0,00698411
0,0097222
0,0122222
-1E+10
-6,143
-5,953
-5,779
-5,779
-5,588
-5,588
-5,391
-5,277
-5,228
-5,164
-5,164
-4,972
-4,972
-4,831
-4,745
-4,678
-4,584
-4,5
-4,413
-4,413
-4,23
-4,143
-4,082
-3,999
-3,929
-3,846
-3,779
-3,725
-3,63
-3,543
-3,49
-3,436
-3,323
-3,278
-3,203
-3,111
-3,037
-2,99
-2,92
-2,86
-2,793
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1,18519
0,855553823
5,133322938
308
0,002777772
0,01666663
-2,734
1E+10
1,144673
0,855553823
7,699984407
334
0,002561538
0,02305385
-2,676
1E+10
1,104862
1,711107646
11,20775508
367
0,004662419
0,03053884
-2,609
1E+10
1,065755
1,711107646
14,88663652
406
0,004214551
0,03666659
-2,541
1E+10
1,027352
2,566661469
18,73662872
446
0,005754846
0,04201038
-2,479
1E+10
0,989654
3,422215292
25,92328084
487
0,007027136
0,05323056
-2,416
1E+10
FDR < 5 % =
proportion of
non differential
genes
969 genes downregulated
5 genes upregulated
RTD
SAM : delta table
Delta Table
delta
# med false pos
3,523156
3,453045
3,383639
3,314937
3,24694
3,179648
3,11306
3,047177
2,981999
2,917525
2,853756
2,790692
2,728332
2,666677
2,605726
2,54548
2,485939
2,427102
2,36897
2,311543
2,25482
2,198802
2,143488
2,088879
2,034975
1,981775
1,92928
1,87749
1,826404
1,776023
1,726346
1,677374
1,629107
1,581545
1,534687
1,488533
1,443085
1,398341
1,354301
1,310966
1,268336
1,226411
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0,855553823
90th perc false
pos
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0,855553823
0,855553823
0,855553823
1,711107646
2,566661469
3,507770674
# called
median FDR
0
2
3
5
5
6
6
8
10
11
12
12
14
14
16
17
19
23
26
29
29
39
46
47
56
62
69
78
79
89
105
109
117
145
150
163
196
217
227
245
264
287
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0,002981024
90th perc
FDR
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0,00436507
0,00394264
0,00376896
0,00698411
0,0097222
0,0122222
cutlo
cuthi
-1E+10
-6,143
-5,953
-5,779
-5,779
-5,588
-5,588
-5,391
-5,277
-5,228
-5,164
-5,164
-4,972
-4,972
-4,831
-4,745
-4,678
-4,584
-4,5
-4,413
-4,413
-4,23
-4,143
-4,082
-3,999
-3,929
-3,846
-3,779
-3,725
-3,63
-3,543
-3,49
-3,436
-3,323
-3,278
-3,203
-3,111
-3,037
-2,99
-2,92
-2,86
-2,793
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1,18519
0,855553823
5,133322938
308
0,002777772
0,01666663 -2,734
1E+10
1,144673
0,855553823
7,699984407
334
0,002561538
0,02305385 -2,676
1E+10
1,104862
1,711107646
11,20775508
367
0,004662419
0,03053884 -2,609
1E+10
1,065755
1,711107646
14,88663652
406
0,004214551
0,03666659 -2,541
1E+10
1,027352
2,566661469
18,73662872
446
0,005754846
0,04201038 -2,479
1E+10
0,989654
3,422215292
25,92328084
487
0,007027136
0,05323056 -2,416
1E+10
delta
# med false pos
0,952661
0,916373
0,880789
0,84591
0,811735
0,778265
0,7455
0,713439
0,682083
0,651432
0,621485
0,592242
0,563705
0,535872
0,508744
0,48232
0,456601
0,431587
0,407277
0,383672
0,360771
0,338575
0,317084
0,296297
0,276215
0,256838
0,238165
0,220197
0,202934
0,186375
0,170521
0,155371
0,140926
0,127186
0,11415
0,101819
0,090193
0,079271
0,069054
0,059541
0,050733
0,04263
0,035232
0,028538
0,022548
0,017263
0,012683
0,008808
0,005637
0,003171
0,001409
0,000352
0
5,133322938
5,988876761
7,699984407
9,411092053
12,40553043
16,68329955
20,96106866
26,94994542
36,36103748
46,19990644
60,31654452
74,4331826
88,54982068
116,3553199
139,0274962
186,9385103
226,2939862
274,6327772
335,8048755
390,9880971
464,5657259
571,0821768
666,904205
770,4262176
900,4703987
1024,95348
1167,830968
1383,858309
1629,830033
1961,784916
2407,956235
2957,649566
3959,503093
4666,19055
5444,316752
6490,231301
7437,75716
8482,816155
9805,930142
10902,75014
11745,47066
12017,53677
12221,15858
12388,41936
12486,80805
12660,48547
12813,62961
12961,21264
13093,39571
13146,01227
13205,47326
13244,82873
13251,24539
90th perc false
pos
32,08326836
45,60101876
57,40766152
74,51873798
97,01980352
118,151983
139,198607
166,9185509
201,3118145
250,8483809
312,5338115
371,9092468
440,6957742
511,5356308
602,3098914
737,5729508
864,3660273
982,2613441
1143,019907
1277,512968
1462,483705
1732,15427
1967,08935
2166,946723
2419,249545
2645,030199
2936,004054
3296,534435
3693,425854
4243,803628
4890,003431
5519,691044
6528,474557
7188,619887
7953,313894
8878,338687
9684,355944
10570,7097
11592,32652
12419,98929
13008,2681
13233,10765
13374,27403
13512,27486
13592,52581
13727,5322
13852,44306
13965,80394
14058,1182
14089,00369
14135,03249
14160,27132
14163,18021
# called
median FDR
541
599
636
677
708
743
774
818
860
930
1008
1070
1136
1237
1323
1472
1595
1693
1842
1948
2089
2351
2531
2683
2890
3065
3275
3594
3958
4435
5019
5708
6834
7568
8429
9599
10515
11604
12939
14023
14858
15091
15260
15404
15480
15628
15774
15915
16041
16075
16126
16155
16160
0,009488582
0,009998125
0,012106894
0,01390117
0,017521936
0,02245397
0,027081484
0,032946144
0,042280276
0,049677319
0,059837842
0,069563722
0,077948786
0,094062506
0,105085031
0,126996271
0,141877107
0,162216643
0,182304493
0,200712576
0,222386657
0,242910326
0,263494352
0,287151032
0,311581453
0,334405703
0,356589609
0,38504683
0,411781211
0,442341582
0,479768128
0,518158649
0,579382952
0,616568519
0,645903043
0,676136191
0,707347329
0,731025177
0,757858424
0,777490561
0,790514918
0,796338001
0,800862293
0,804233923
0,806641347
0,810115528
0,812325955
0,814402302
0,816245602
0,817792365
0,818893294
0,819859408
0,820002809
www.diogenes-eu.org
90th perc FDR cutlo
0,05930364
0,07612858
0,09026362
0,11007199
0,13703362
0,15902017
0,17984316
0,20405691
0,23408351
0,26972944
0,31005338
0,34757874
0,38793642
0,41352921
0,45526069
0,50106858
0,54192227
0,58018981
0,62053198
0,65580748
0,70008794
0,7367734
0,77719848
0,80765812
0,83711057
0,86297886
0,89648979
0,91723273
0,93315459
0,95688921
0,97429835
0,96700964
0,95529332
0,94987049
0,94356553
0,92492329
0,92100389
0,91095396
0,89592136
0,88568704
0,875506
0,87688739
0,87642687
0,8771926
0,87807014
0,87839341
0,87818201
0,8775246
0,87638665
0,87645435
0,8765368
0,87652562
0,87643442
-2,351
-2,284
-2,231
-2,179
-2,132
-2,084
-2,039
-1,99
-1,943
-1,891
-1,836
-1,786
-1,743
-1,693
-1,646
-1,593
-1,544
-1,502
-1,453
-1,413
-1,368
-1,317
-1,275
-1,236
-1,188
-1,147
-1,108
-1,065
-1,026
-0,979
-0,926
-0,881
-0,825
-0,783
-0,744
-0,708
-0,666
-0,631
-0,602
-0,57
-0,543
-0,508
-0,48
-0,457
-0,442
-0,419
-0,397
-0,377
-0,359
-0,353
-0,345
-0,341
-0,34
cuthi
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
1E+10
3,5500644
3,1950525
3,1950525
3,1950525
2,7635623
2,5868176
2,5054882
2,3087747
2,2369046
2,1696993
2,089767
2,0287648
1,9607458
1,8401803
1,772953
1,7172366
1,6695726
1,6252992
1,5628222
1,481335
1,3879799
1,2931184
1,1975641
1,085506
0,9311183
0,8379125
0,7312739
0,5929879
0,4991773
0,3846652
0,2438373
0,1358228
0,0522468
0,0503664
0,0503664
0,0503664
0,0503664
0,0503664
0,0503664
0,0503664
0,0503664
0,0503664
0,0503664
0,0503664
0,0503664
FDR = 10,51%
1322 genes downregulated
72 genes upregulated
Calorie restriction effect on gene expression profile
observed
Overexpressed genes
2500
Underexpressed genes
Number of significantly regulated genes
depending on the false discovery rate level
Number of significant genes
Expected (random)
up
down
2000
1500
1000
500
0
1
5
10
15
False Discovery Rate (%)
20
30
SAM : list of significant gene
Delta = 0.651, FDR < 5%
RTD
Positive genes (5)
1
2
3
4
5
Row
152
75
28
184
87
Gene ID
18188
36392
14986
31808
10028
CLID
IMAGE:81434
IMAGE:344970
IMAGE:115277
IMAGE:284524
IMAGE:122321
Score(d)
3,55006443
3,298706978
3,208703717
3,195052469
3,074964182
Numerator(r)
0,001591828
0,002033256
0,002572328
0,001282799
0,002042801
Denominator(s+s0)
0,000448394
0,00061638
0,000801672
0,000401496
0,000664333
q-value(%)
4,22802761
4,96773188
4,96773188
4,96773188
4,96773188
Negative genes (969)
1
2
3
4
5
6
7
8
965
966
967
968
969
Row
110
74
136
15
190
146
60
77
4541
6717
4616
####
6316
Gene ID
34872
36264
11284
8278
38283
11895
26169
39139
17765
30370
18562
31786
16651
CLID
IMAGE:123474
IMAGE:126887
IMAGE:1584541
IMAGE:502333
IMAGE:813387
IMAGE:206272
IMAGE:23831
IMAGE:813387
IMAGE:767988
IMAGE:153883
IMAGE:240295
IMAGE:132748
IMAGE:2568305
Score(d)
-6,29361535
-6,142716806
-5,953441535
-5,852904282
-5,779443418
-5,588241215
-5,432269789
-5,391369137
Numerator(r)
-0,011385421
-0,003515042
-0,002871662
-0,006631314
-0,003262757
-0,004347608
-0,004945055
-0,00286869
Denominator(s+s0)
0,001809043
0,000572229
0,000482353
0,001132996
0,000564545
0,000777992
0,000910311
0,000532089
q-value(%)
0
0
0
0
0
0
0
0
-1,86490263
-1,864846369
-1,864565522
-1,864474282
-1,863583984
-0,000983864
-0,001324057
-0,000857983
-0,001307669
-0,001210904
0,000527569
0,000710009
0,000460152
0,000701361
0,000649772
4,96773188
4,96773188
4,96773188
4,96773188
4,96773188
www.diogenes-eu.org
SAM : list of significant gene
Delta = 0.651, FDR < 5%
RTD
Positive genes (5)
1
2
3
4
5
Row
152
75
28
184
87
Gene ID
18188
36392
14986
31808
10028
CLID
IMAGE:81434
IMAGE:344970
IMAGE:115277
IMAGE:284524
IMAGE:122321
Score(d)
3,55006443
3,298706978
3,208703717
3,195052469
3,074964182
Numerator(r)
0,001591828
0,002033256
0,002572328
0,001282799
0,002042801
Denominator(s+s0)
0,000448394
0,00061638
0,000801672
0,000401496
0,000664333
q-value(%)
4,22802761
4,96773188
4,96773188
4,96773188
4,96773188
Gene Symbol
Mean Fold-Change
ALOX12
1.13001191395484
ITGA4
1.18690578270973
GPR65
1.24993574814629
LRP1B
1.09511788559594
CSPG2
1.18323402304505
Negative genes (969)
1
2
3
4
5
6
7
8
965
966
967
968
969
Row
110
74
136
15
190
146
60
77
4541
6717
4616
####
6316
Gene ID
34872
36264
11284
8278
38283
11895
26169
39139
17765
30370
18562
31786
16651
CLID
IMAGE:123474
IMAGE:126887
IMAGE:1584541
IMAGE:502333
IMAGE:813387
IMAGE:206272
IMAGE:23831
IMAGE:813387
IMAGE:767988
IMAGE:153883
IMAGE:240295
IMAGE:132748
IMAGE:2568305
Score(d)
-6,29361535
-6,142716806
-5,953441535
-5,852904282
-5,779443418
-5,588241215
-5,432269789
-5,391369137
Numerator(r)
-0,011385421
-0,003515042
-0,002871662
-0,006631314
-0,003262757
-0,004347608
-0,004945055
-0,00286869
Denominator(s+s0)
0,001809043
0,000572229
0,000482353
0,001132996
0,000564545
0,000777992
0,000910311
0,000532089
q-value(%)
0
0
0
0
0
0
0
0
Gene Symbol
Mean Fold-Change
SCD
ACOX1
ATP8A2
0.573156854700039
NCOA3
NQO1
0.697308449088142
ME1
ALDOC
0.775342788540698
NQO1
0.830387636013746
-1,86490263
-1,864846369
-1,864565522
-1,864474282
-1,863583984
-0,000983864
-0,001324057
-0,000857983
-0,001307669
-0,001210904
0,000527569
0,000710009
0,000460152
0,000701361
0,000649772
4,96773188
4,96773188
4,96773188
4,96773188
4,96773188
ELL2
KIAA1881
FNDC3B
DOCK9
BCAP31
0.943850733965601
0.938937459267326
0.948093296468166
0.956614026485153
0.953308970534193
www.diogenes-eu.org
0.802848550620883
0.82851984305876
0.830387636013746
0.766582052707411
RTD
ONTOLOGY
Ontology
: PANTHER
= lists with gene symbol
www.diogenes-eu.org
RTD
Ontology : biological process
(PANTHER) DOWN
Number of
genes in
this
biological
process
DOWN:
843 Gene symbols matched / 1322 Gene symbols
15 Biological processes defined with P-value < 0.5%
www.diogenes-eu.org
RTD
Ontology : biological process
(PANTHER) UP
UP:
55 Gene symbols matched / 72 Gene Symbols
4 Biological processes defined with P-value < 5%
www.diogenes-eu.org
Ontology : Pathway diagram
(PANTHER)
Glycolysis :
6 down regulated genes
Microarray workflow process: major phases of microarray analysis
Short summary - end
•
•
•
The typical result of a microarray experiment
A list of differentially expressed genes
Goal
Find genes or gene sets associated with the response to treatment
Question
How to analyze microarray data?
•
Biological theory is not about isolated genes
About pathways
About biological processes
About areas of the genome
About sets of related genes
•
Annotation database Links genes to annotation !
Gene Ontology
KEGG
creates sets of genes: GSEA
Chromosomal location
Some conclusions
• For years, scientists studied one gene or protein at a time.
• Features were thus studied in isolation from the larger context of
other ones.
• Omics aims to study cells, tissue or organisms as a whole.
• Omics is based on high-throughput techniques allowing a wide
picture of gene characteristics.
Conclusions II
• high-throughput technologies are reshaping biology.
• symptomatic characteristic : new scientific journals more adapted to the
new approaches
–
–
–
–
–
Genomics, 1987
Physiological Genomics , 1999
Functional & Integrative Genomics , 2000
Proteomics , 2001
Metabolomics and PLoS Computational Biology , 2005.
• names of these Journals indicate the evolution of Science from Genes
to Metabolites via transcripts and proteins.
• Omics will drive enormous changes in biology
Conclusions III
• Omic is as much as technology allows
• Powerful changes:
– Drug
– Illness
•
•
•
•
Significant but subtle changes: diet
My Omic better than your Omic?
New area: metabolomics
Integrative biology is the challenge to face in the next
decades.
Abbreviations used
HOMA
Homeostasis model assessment
IVT
In vitro transcription
GPR
Genepix results (file format .gpr)
KEGG
Kyoto Encyclopedia of Genes and Genomes
GSEA
Gene Set Enrichment Analysis