Applications of microarrays

Gene Target Discovery Pharmacology and Toxicology

• Ex vivo analysis of diseases • Drug´s activity and toxicity
• Cell culture modells • non-responder
• Pathophysiology of diseases • Parameter for clinical studies
• therapeutic und diagnostic targets • Reduction of animal studies

Diagnostics
• Disease-specific gene expression
• Monitoring in therapy
• Screening of mutations- und polymorphisms
Production and analysis of cDNA/oligonucleotide arrays

Affymetrix Affymetrix Pipettier-

GMS 417 Arrayer 428 Scanner roboter
Hybridisation of arrays with two fluorophores

Cells Poly (A)+ RNA

or Total RNA
R T -P C R

Cy3
Hybridisation

Cy5

Scan
Data analysis
Identification of genes differentially expressed in patients
with isolated Lp(a) hyperlipidemia

M o n o c y te s
10.000 cDNAs

C o n tro l
114 genes were
different

P a t ie n t s w ith L p ( a )
h y p e r lip d e m ia

10.000 cDNAs
109 genes:
C o n tro l Interindividual
variance

5 genes:
P a t ie n t s w ith L p ( a ) Up- or downregulated
h y p e r lip d e m ia
in patients
Synthesis of Affymetrix Microarrays
 Hybridisation of Affymetrix Microarrays

Biotin - labeled
Cells cRNA transcript
In-vitro B
+ B
Poly (A) transcription B
B
B B B
RNA
Or Biotin-UTP
Total RNA Fragment
cDNA Biotin-CTP
heat, Mg2+

B
B
B
Hybridize B
B

Wash & Stain

(16 hours)
Scan (75 minutes)

(8 minutes)
 GeneChip® Instrument System

Fluidics Scanner made by Computer

Station Hewlett-Packard Workstation
 ®
GeneChip Probe Array
 Analysis of microarray data
Human Genome U95 Set: Five Gene Chip Arrays, 63,000 probe sets, 54,000 clusters
HG-U95Av2 array: 10,000 full length genes Array B through E: EST clusters
 Analysis of microarray data

P ro b e s e t G ene nam e Load Load

20 91_ a t w i n g le s s - t y p e M M T V in t e g r a t i o n s it e f a m il y , m e m b e r 4 N C N C N C
38 198 _ at H o m o s a p ie n s m R N A f u l l le n g t h i n s e r t c D N A c l o n e E U R O I M A G E 5 6 6 4 4 3 N C N C N C
38 081 _ at le u k o t r ie n e A 4 h y d r o la s e N C N C N C
33 729 _ at s o l u t e c a r r ie r f a m i l y 2 5 ( m it o c h o n d r i a l c a r r ie r , b r a i n ) , m e m b e r 1 4 N C N C N C

33 825 _ at s e r i n e ( o r c y s t e in e ) p r o t e i n a s e i n h ib it o r , c la d e A ( a lp h a - 1 a n t ip r o t e i n a s e , a n t it r y p s i n ) , m e m b e r 3 N C D N C
32 851 _ at C U G t r i p l e t r e p e a t , R N A - b i n d i n g p r o t e in 2 N C D N C
16 51_ a t u b iq u i t i n c a r r i e r p r o t e i n E 2 - C N C D N C
33 166 _ at T N F R S F 1 A - a s s o c i a t e d v i a d e a t h d o m a in N C NC N C
h y d r o x y a c y l- C o e n z y m e A d e h y d r o g e n a s e / 3 - k e t o a c y l- C o e n z y m e A t h io la s e / e n o y l- C o e n z y m e A
39 741 _ at h y d r a t a s e ( t r i f u n c t i o n a l p r o t e in ) , b e t a s u b u n i t N C NC N C
98 2_a t m i n i c h r o m o s o m e m a in t e n a n c e d e f ic i e n t ( S . c e r e v is i a e ) 5 ( c e l l d iv i s io n c y c le 4 6 ) N C NC N C
35 371 _ at c e l l d iv i s io n c y c le 4 - l ik e N C D N C
39 999 _ at c a s p a s e 1 0 , a p o p t o s is - r e la t e d c y s t e in e p r o t e a s e N C NC D
39 372 _ at a c c e s s o r y p r o t e in s B A P 3 1 / B A P 2 9 D D DD
36 708 _ at c a r b o x y p e p t id a s e M N C D N C
40 505 _ at u b iq u i t i n - c o n j u g a t i n g e n z y m e E 2 L 6 N C NC N C
36 990 _ at u b iq u i t i n c a r b o x y l- t e r m i n a l e s t e r a s e L 1 ( u b i q u it i n t h io le s t e r a s e ) N C NC D
12 94_ a t u b iq u i t i n - a c t i v a t i n g e n z y m e E 1 - l ik e N C NC N C
m e t h y le n e t e t r a h y d r o f o la t e d e h y d r o g e n a s e ( N A D P + d e p e n d e n t ) , m e t h e n y lt e t r a h y d r o f o la t e
67 3_a t c y c l o h y d r o l a s e , f o r m y lt e t r a h y d r o f o la t e s y n t h e t a s e N C NC N C
39 373 _ at H o m o s a p ie n s c l o n e 2 3 7 1 6 m R N A s e q u e n c e DD D DD
37 700 _ at b l e o m y c i n h y d r o la s e N C D N C
41 722 _ at n i c o t i n a m i d e n u c l e o t id e t r a n s h y d r o g e n a s e D D N C
40 214 _ at a c c e s s o r y p r o t e in s B A P 3 1 / B A P 2 9 N C N C N C
35 662 _ at c a s p a s e 6 , a p o p t o s i s - r e la t e d c y s t e in e p r o t e a s e N C D N C
39 4_a t b l e o m y c i n h y d r o la s e N C N C N C
38 368 _ at d U T P p y r o p h o s p h a ta s e N C D N C
37 944 _ at G T P c y c lo h y d r o l a s e 1 ( d o p a - r e s p o n s i v e d y s t o n ia ) N C D I
38 790 _ at e p o x i d e h y d r o l a s e 1 , m ic r o s o m a l ( x e n o b i o t i c ) N C N C N C
36 830 _ at m i t o c h o n d r ia l i n t e r m e d i a t e p e p t i d a s e N C N C N C
34 742 _ at B C L 2 - l ik e 1 N C N C N C
32 747 _ at a l d e h y d e d e h y d r o g e n a s e 2 , m i t o c h o n d r ia l N C N C N C
36 876 _ at f u m a r y la c e t o a c e t a t e N C N C N C
32 664 _ at r ib o n u c le a s e , R N a s e A f a m i ly , 4 N C N C N C
34 666 _ at s u p e r o x i d e d i s m u t a s e 2 , m i t o c h o n d r ia l D D N C
40 684 _ at G T P c y c lo h y d r o l a s e I f e e d b a c k r e g u la t o r y p r o t e i n II II
37 647 _ at a c y lo x y a c y l h y d r o l a s e ( n e u t r o p h i l) D D N C
33 862 _ at p h o s p h a t i d i c a c id p h o s p h a t a s e t y p e 2 B N C NC I
 Verification with Taqman or LightCycler

14

12

E-LDL
E-LDL
10 E-LDL
E-LDL

8 HDL

M-CSF

HDL
6

HDL
M-CSF

M-CSF
M-CSF

0
1 1
Donor 2 2
Donor 3 3
Donor
 Analysis of microarray data
Human genome Set U133 (HG-U133)
2 Arrays, 45,000 probe sets, 39,000 transcripts, 33,000 human genes
HG-U133A: HG-U95Av2, HG-U133B: EST-clusters

ID D e s c rip tio ns U nig e ne

Lipid related genes

FA Homo sapiens, adipose differentiation-related protein, clone MGC:10598, mRNA, complete cds. Hs.3416 ++ 3688,3 ++ 0,9 ++ 0,4
Apolipoproteins and related
FC Consensus includes gb:AI861942 Hs.213289 - 2 - 1,3 + 19,4
FC low density lipoprotein receptor (familial hypercholesterolemia) (LDLR), mRNA. Hs.213289 + 28,8 + 1,3 ++ 8,1
FC Consensus includes gb:AA885297 Hs.323567 ++ 773 ++ 0,5 ++ 0,6
FC CD36 antigen (collagen type I receptor, thrombospondin receptor)-like 2 (lysosomal integral membrane
Hs.323567
protein II) ++
(CD36L2),
422,3mRNA.
++ 0,6 ++ 0,6
Phospholipases
FDB phospholipase C, epsilon (PLCE), mRNA. Hs.153322 + 26,9 + 0,9 + 0,5
Phospholipid kinases
FDC diacylglycerol kinase, alpha (80kD) (DGKA), mRNA. Hs.172690 + 52,2 + 2,0 ++ 1,2
FDC Human phosphatidylinositol 3-kinase delta catalytic subunit mRNA, complete cds. Hs.162808 ++ 66,6 + 0,9 + 1,0
FDC Human 68 kDa type I phosphatidylinositol-4-phosphate 5-kinase alpha mRNA, clone PIP5KIa3, complete
Hs.149255
cds. - 52,5 - 0,5 + 0,9
Phospholipid phosphatases
FDD Consensus includes gb:BE732345 Hs.41569 + 319,1 + 0,5 + 1,7
Sphingolipid metabolism
FDE Human serine palmitoyltransferase (LCB2) mRNA, partial cds. Hs.59403 + 20,6 + 1,2 + 1,2
Fatty acid synthesis
FEA Fatty acid desaturase 1 Hs.132898 + 177,3 + 1,1 + 5,1
NI mRNA; cDNA DKFZp762M2311 (from clone DKFZp762M2311); complete cds. Hs.132898 + 290,9 + 0,9 ++ 2,5
FEA delta-6 fatty acid desaturase (FADSD6), mRNA. Hs.184641 + 62,4 ++ 0,9 + 9,4
FEA Consensus includes gb:AA678241 Hs.119597 + 661,9 ++ 0,9 ++ 2,0
FEA Scd mRNA for stearoyl-CoA desaturase, complete cds. Hs.119597 ++ 1003,8 ++ 1,1 ++ 2,1
FEA PRO0998 mRNA, complete cds. Hs.119597 + 280,5 + 0,7 + 2,0
Cholesterol sterol metabolism
FEC Consensus includes gb:AL518627 Hs.11899 + 50,9 + 0,9 + 2,8
FEC Consensus includes gb:AI189359 Hs.3828 - 11,8 - 1,1 + 2,9
FEC Consensus includes gb:AA872727 Hs.48876 + 113,4 ++ 1,2 ++ 4,3
FEC Homo sapiens, farnesyl-diphosphate farnesyltransferase 1, clone MGC:2200, mRNA, complete cds. Hs.48876 ++ 153,3 ++ 1,1 ++ 3,4
FEC squalene epoxidase (ERG1) mRNA, complete cds. Hs.71465 ++ 77,2 ++ 1,0 ++ 5,6
FEC Consensus includes gb:BF979497 Hs.71465 - 17,1 - 0,7 + 3,2
DE Consensus includes gb:AW150953 Hs.11806 - 53,6 - 1,2 + 4,4
FEC 7-dehydrocholesterol reductase (DHCR7), mRNA. Hs.11806 - 36,7 - 0,9 + 4,3
FEC cytochrome P450, 51 (lanosterol 14-alpha-demethylase) (CYP51), mRNA. Hs.226213 + 29,2 ++ 1,2 ++ 3,6
FEC Human lanosterol 14-alpha demethylase (CYP51P2) processed pseudogene, complete cds Hs.226213 ++ 119,8 + 0,8 ++ 3,0
FEC acyl-coenzyme A: cholesterol acyltransferase mRNA, complete cds. Hs.14553 ++ 203,7 ++ 0,9 + 0,4