introduction to dna microarrays dtu - january 2007 - hanne jarmer

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