introduction to dna microarrays dtu - january 2007 - hanne jarmer
TRANSCRIPT
Introduction to
DNA microarrays
DTU - January 2007 - Hanne Jarmer
Microarrays - The Concept
Measure the level of transcript from a
very large number of genes in one go
CELL
RNA
Why?
RNA
How?
gene specific DNA probeslabeled target
gene
mRNA
Microarrays - The Technologies
Stanford-type Microarrays
High-density
Stanford-type Microarrays
Stanford-type Microarrays
Coating glass slides
Deposition of probes
Post-processing
Hybridization
Spotting - Mechanical deposition of probes
16-pin microarrayer
Microarrayer
mRNAmRNA
cDNAcDNA
Cy3-cDNACy5-cDNA
SAMPLE CONTROL
Stanford
microarrays
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
• 280-500 K features to play with
TTT
T
T
TT
T
T
T
A
A
AA
A
A
A
AAA
Photolithographyin situ synthesis
Spacers bound to surface with photolabile protection groups
Mask #1Mask #2
Sample Preparation- Eberwine
RNA
T7
dsDNA
T7 pol
SAMPLE
ssDNA
+ Reverse Transcriptase
+ RNase H+ Polymerase clean up dsDNA
+ Biotin-labeled nucleotides
aRNA
42 C2 h
16 C2 h
37 C6 h
70 C10 min
Detection of Biotin (Affymetrix)
Streptavidin Phycoerythrim = SAPE ( )
anti-SAPE IgG
biotinylated anti-anti IgG
The Affymetrix GeneChip®
A gene is represented like this:
- Perfect Match (PM)- MisMatch (MM)
PMMM
PM: CGATCAATTGCACTATGTCATTTCT MM: CGATCAATTGCAGTATGTCATTTCT
NimbleGen
• 385,000 to 2.1 mill features
• Long probes (up to 70 nt)
• Service:
-labelling
-scanning
-image analysis
Photolithography - Micromirrors
Analysis of Data
Normalization:Linear or non-linear
Is it worth it?
Nu
mb
er
of
kn
ow
n p
osit
ives
Number of significantly affected genes
Qspline normalization
Linear normalization
Known positives versus the total number of significantly affected genes at 5 different cutoffs in the
TnrA experiment
Analysis of Data
Normalization:Linear or non-linear
Statistical test:student’s t-testANalysis Of VAriance
(ANOVA)Analysis:
Principal Component Analysis (PCA) Clustering and visualization
Tiling arrays
Tiling arrays are used for determation of genes, ncRNAs, TF-binding sites, ...
Regulatory T Cells and Allergy
Hanne Jarmer (CBS)
Type I Hypersensitivity
A normal reaction against parasitesTypical allergens:
- pollen- penicillin- nuts- milk- bee venom- mold spores
Early Last Century
“Hmm..., maybe we can make a vaccine?”
Portuguese Man of War
Poul Portier and Charles Richet observed:
Early last century
Early Last Century
1. time: No reaction
2. time: Got sick died
Immediate Hypersensitivity
anaphylaxis
Richet got the Nobel Prize
The Mechanism
The Regulation
- presentation (concentration/mode)
- TH1, TH2 and TReg
Several factors are important:
- genetics
The Regulation
The Naive T cell will differentiate into different subsets induced by different cytokines
Naive T cell
Cytokines
TH1 TH2 TReg
TC
The Regulation
NaiveT cell
TH1 TH2 TReg
IL-12
IL-4IL-10 ?
CD4+ T cells - The normal state
TH1 TH2
TReg
- Keeping the balance
The allergic state
TH1
TH2
TReg
- Out of balance
Three types of TReg cells
IL-10 TGF-
CD25
CD25 = IL-2 receptor (IL2RA)
TReg1 TReg2 TReg3
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 inImmuno Specific Therapy (SIT) - graft tolerance
... or the other way around ... in vaccines or cancer treatment
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
The Microarray Experiments
normal allergicnormalCD4+CD25+
normalCD4+
allergic CD4+CD25+
allergicCD4+
Blood from 5 normal and 5 allergic patients
20 microarrays
The Microarray Experiments
20 microarrays
2-way ANOVA
NormalCD4+CD25+
NormalCD4+
Allergic CD4+CD25+
AllergicCD4+
2
2
1 1
2-way ANOVA
The Regulatory T cell signal
Nature Immunology 6, 345 - 352 (2005)Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-selfby Shimon Sakaguch
Natural Treg cells
Maybe ...
TReg
X
(TCR MHC-II)APC
Allergic TReg: Less XFewer active TReg cellsNo tolerance
CTLA-4 B7
Kristine Dahlin and Thomas Jensen
Thanks to
ALK-abello
Liu Anting, PhD from ALK