1. DTU - January 2007 - Hanne Jarmer
Introduction to
DNA microarrays
DTU - January Hanne Jarmer

2. Microarrays - The Concept
Measure the level of transcript from a very large number of genes in one go
RNA
CELL

3. Why?
RNA

4. gene specific DNA probes
How?
gene
mRNA
gene specific DNA probes
labeled target

5. Microarrays - The Technologies
Stanford-type Microarrays
High-density

6. Stanford-type Microarrays

7. Stanford-type Microarrays
Coating glass slides
Deposition of probes
Post-processing
Hybridization

8. Spotting - Mechanical deposition of probes

9. 16-pin microarrayer

11. Microarrayer

12. Stanford microarrays
SAMPLE
CONTROL
mRNA
cDNA
Cy3-cDNA
Cy5-cDNA

13. Affymetrix GeneChip® oligonucleotide array
• 11 to 20 oligonucleotide probes for each gene
On-chip synthesis of 25 mers
~20,000 genes per chip
good quality data
K features to play with

14. Photolithography Mask #2 Mask #1
in situ synthesis T A
Mask #2
Mask #1 T A
Spacers bound to surface with photolabile protection groups

15. Sample Preparation - Eberwine
RNA
42 C
2 h
ssDNA
+ Reverse Transcriptase
70 C
10 min T7
+ RNase H
+ Polymerase
16 C
2 h
T7 pol
clean up dsDNA
dsDNA
37 C
6 h
+ Biotin-labeled nucleotides
aRNA

16. Detection of Biotin (Affymetrix)
Streptavidin Phycoerythrim = SAPE ( )
anti-SAPE IgG
biotinylated anti-anti IgG

17. The Affymetrix GeneChip®
A gene is represented like this: PM MM
- Perfect Match (PM)
- MisMatch (MM)
PM: CGATCAATTGCACTATGTCATTTCT
MM: CGATCAATTGCAGTATGTCATTTCT

18. NimbleGen 385,000 to 2.1 mill features Long probes (up to 70 nt)
Service:
labelling
scanning
image analysis

19. Photolithography - Micromirrors

20. Analysis of Data
Normalization:
Linear or non-linear

21. Is it worth it?
Known positives versus the total number of significantly affected genes at 5 different cutoffs in the TnrA experiment
Number of known positives
Qspline normalization
Linear normalization
Number of significantly affected genes

22. Analysis of Data Normalization: Linear or non-linear Statistical test:
student’s t-test
ANalysis Of VAriance (ANOVA)
Analysis:
Principal Component Analysis (PCA)
Clustering and visualization

23. Tiling arrays
Tiling arrays are used for determation of genes, ncRNAs, TF-binding sites, ...

24. Regulatory T Cells and Allergy
Hanne Jarmer (CBS)

25. Type I Hypersensitivity
A normal reaction against parasites
Typical allergens:
- pollen
- penicillin
- nuts
- milk
- bee venom
- mold spores

26. Early Last Century Poul Portier and Charles Richet observed:
Portuguese Man of War
“Hmm..., maybe we can make a vaccine?”

27. Early last century

28. Immediate Hypersensitivity Richet got the Nobel Prize
Early Last Century
1. time: No reaction
2. time: Got sick  died
Immediate Hypersensitivity
anaphylaxis
Richet got the Nobel Prize

29. The Mechanism

30. The Regulation
Several factors are important:
- genetics
presentation (concentration/mode)
- TH1, TH2 and TReg

31. The Regulation TC TH1 TH2 TReg
The Naive T cell will differentiate into different subsets induced by different cytokines
Naive T cell
Cytokines
TH1 TH2 TReg TC

32. The Regulation
Naive
T cell
IL-12
IL-4
IL-10 ?
TH1
TReg
TH2

33. CD4+ T cells - The normal state
- Keeping the balance
TH1
TH2
TReg

34. The allergic state
- Out of balance
TReg
TH1
TH2

35. Three types of TReg cells
CD25
TReg1
TReg2
TReg3
IL-10
TGF-
CD25 = IL-2 receptor  (IL2RA)

36. CD25+ TReg cells Low proliferative capacity
Immunoregulatory properties
T cell homeostasis
Prevents autoimmunity
Antigen specific, cell-cell adhesion
Induction of tolerance ... but how?
Potential use in
Immuno Specific Therapy (SIT) - graft tolerance
... or the other way around ...
in vaccines or cancer treatment

37. Microarray Project
The goal: Investigate the mechanism
 find cure for allergy or an effective SIT
The plan: Find the responsible genes by the
use of carefully designed microarray experiments

38. The Microarray Experiments
Blood from 5 normal and 5 allergic patients
normal
allergic
normal
CD4+CD25+
CD4+
allergic
20 microarrays

39. The Microarray Experiments
20 microarrays

40. 1 2-way ANOVA 2 Normal Allergic Normal Allergic CD4+CD25+ CD4+CD25+

41. 2-way ANOVA

42. The Regulatory T cell signal
Natural Treg cells
Nature Immunology  6, (2005)
Naturally arising Foxp3-expressing CD25+CD4+ regulatory
T cells in immunological tolerance to self and non-self
by Shimon Sakaguch

43. Fewer active TReg cells No tolerance
Maybe ...
CTLA-4 B7 X
APC
TReg
(TCR
MHC-II)
Allergic TReg: Less X
Fewer active TReg cells
No tolerance

44. Thanks to Liu Anting, PhD from ALK ALK-abello
Kristine Dahlin and Thomas Jensen